Single cell analysis

0001The present invention concerns processes for barcoding nucleic acids from single cells and processes for genotyping single cells having a phenotype of interest.

0002Methods for analyzing multiple parameters of single cells in populations are of interest in a variety of contexts. In particular, the ability to analyze nucleic acids, optionally in combination with one or more other parameters, on a single-cell basis within a cell population is of broad interest to commercial and academic laboratories. Moreover, it is often necessary for this analysis to be performed in real-time in order to reveal the dynamic behavior of biological and biochemical processes.

0003Thus, technologies that can isolate, detect, and quantify individual components of a heterogeneous mixture in a highly parallel fashion are needed to meet these challenges. Conventional high-throughput platforms such as high-density microwell plates with robotic dispensing systems have been developed and widely used for high-throughput analysis, such as drug screening. However, they require expensive and bulky robotic machinery and suffer from sample evaporation and comparably large reaction volumes, which can waste precious biological samples or reagents

0004Recently, microfabricated devices have emerged as a powerful experimental platform for performing a diverse range of biological and chemical assays in a high-throughput manner. These technologies often permit high-throughput analysis of a complex sample by partitioning a bulk solution into many isolated pico to nanoliter-sized compartments, or microreactors. However, post-analysis retrieval of individual samples is difficult to achieve. Furthermore, mixing of reagents in these devices either requires complex architecture or is often done in bulk before compartmentalization, which may prevent initial reaction products from co-localizing with their initiating target.

0005One approach is to compartmentalize reactions into discrete micron-sized droplets surrounded by an immiscible carrier fluid. Droplet-based microfluidics provides precise control over mixing of fluids, minimizes waste of precious reagents, and reduces evaporation and adsorption of molecules at the device walls.

0006<nplcit id="ncit0001" npl-type="s"><text>White et al. (Proc Natl Acad Sci USA. 2011 Aug 23;108(34):13999-4004</text></nplcit>) describe a microfluidic device capable of performing RT-qPCR measurements of gene expression from hundreds of single cells per run, executing single-cell processing, including cell capture, cell lysis, reverse transcription, and quantitative PCR. However, incited to try smaller reaction volumes, <nplcit id="ncit0002" npl-type="s"><text>White et al. (Proc Natl Acad Sci USA. 2011 Aug 23; 108(34):13999-4004</text></nplcit>) performed 300 parallel RT-qPCR and demonstrated that RT is inhibited in volumes that are smaller than 5nL in the reaction conditions tested. The researchers thus performed the RT reaction in 67nL per cell and further claimed that the combination of RT and qPCR in a single reaction precludes large-scale transcriptome analysis and/or unbiased amplification.

0007Furthermore, a method using a simple axisymmetric flow-focusing device has been described for single cell mRNA capture by <nplcit id="ncit0003" npl-type="s"><text>DeKosky et al. (Nat Med. 2015 Jan; 21(1):86-91</text></nplcit>.). The RT and PCR reaction of the method take place in an emulsion. This process has proven to be efficient, yet requiring performing sequential 3 steps process.

0008Along the same line, <nplcit id="ncit0004" npl-type="s"><text>Eastburn et al. (Anal Chem. 2013 Aug 20;85(16):8016-21</text></nplcit>) performed RT in small droplet volume, yet in a time consuming 3-4 steps process. They mentioned also RT inhibition in small volume from cell proteinase. To overcome this problem, they treat the cells with proteinase, dilute cell lysate, split drops, and pico-inject RT-PCR reagents.

0009<nplcit id="ncit0005" npl-type="s"><text>Rotem et al. (PLoS One. 2015 May 22; 10(5):e0116328</text></nplcit>) encapsulate oligo in droplet population and fuse them to droplet containing cells while pico-injecting the RT enzymes and buffers. This 3 step process produces ∼100pL droplets allowing encapsulation of 100.000 cells in 3h. Although this method relies on single cell cDNA labeling, the transcriptomic sequence data comes from an aggregate of multiple phenotypically and genotypically uncorrelated cells.

0010<nplcit id="ncit0006" npl-type="s"><text>Macosko et al. (Cell. 2015 May 21; 161(5):1202-14</text></nplcit>) used droplet-based microfluidics to encapsulate cells together with lysis reagents and barcoded beads to capture mRNA in 1nL drops. The beads are, however, used off chip to perform the conversion of the mRNA captured on beads into cDNA and they do not load into most of the formed droplet thus precluding analysis of rare cell populations. The third step of the method described by Macosko et al. is then devoted to library preparation and amplification.

0011The international patent application <patcit id="pcit0001" dnum="WO2015164212A"><text>WO2015/164212</text></patcit> refers to a method for encapsulating and barcoding single cell nucleic acids. <patcit id="pcit0002" dnum="WO2015164212A"><text>WO2015/164212</text></patcit> discloses that RT of mRNA is strongly inhibited in volumes that are smaller than 3nL (Example 4 of <patcit id="pcit0003" dnum="WO2015164212A"><text>WO2015/164212</text></patcit>).

0012<nplcit id="ncit0007" npl-type="s"><text>Labanieh et al. (Micromachines 2015, 6(10):1469-1482</text></nplcit>) describes a microfluidic process using a microfluidic device to trap, analyse, and recover droplets using buoyancy. However, Labanieh et al. does not specify the analysis of single cell nucleic acids wherein the nucleic acids are reverse transcribed and therefore the genotype of single cells cannot be obtained using this method.

0013The patent application <patcit id="pcit0004" dnum="US20130323732A"><text>US2013/0323732</text></patcit> refers to methods and devices for assaying single cells and barcoding single cell cDNA. <patcit id="pcit0005" dnum="US20130323732A"><text>US2013/0323732</text></patcit> does not disclose a method wherein droplets are fused within a microfluidic chip.

0014Contrary to this, the inventors succeeded in developing an on chip microfluidic process for barcoding single cell nucleic acids, wherein single cell droplets are captured in individual compartments. The single cell droplets are further fused with droplets providing the reaction mixture for reverse transcription.

0015As mentioned above, the on chip process developed by the inventors allows accessing the phenotype of single cells. The inventors therefore further developed a process uniquely coupling phenotype information (protein expression level, cellular pathway activation/activity, ion channel/GPCR activities) with genotypic or epigenetic information, thus allowing determining the genotype of a single cell having a phenotype of interest.

0016The ability to image the array at each step in the process allows obtaining more quantitative data per single cell than with conventional methods and allows generating kinetic data during the phenotypic screen which can be then linked with the genotype of a particular single cell.

0017This process differs from the so far known prior art, such as Rotem et al., because in Rotem et al., for example, genomic information is obtained by sequencing an aggregate of multiple phenotypically and genotypically uncorrelated cells. Their method therefore does not disclose coupling phenotype information to the genotype of each single cell.

0018The international patent application <patcit id="pcit0006" dnum="WO2016145409A1"><text>WO2016145409 A1</text></patcit> discloses a method wherein phenotypes and genotypes of a protein of interest may be correlated. In this method, different to the method of the inventors, a barcode is linked to, for example, a protein of interest or its binding partner, and at the same time to the nucleic acid encoding said protein of interest. The nucleic acids are then reverse transcribed to obtain barcoded cDNA, the barcoded cDNA is then sequenced. The protein of interest is in parallel phenotyped, by breaking the emulsion and performing a phenotypic assay, such as ELISA, or an essay using affinity columns. For the proteins that show the desired phenotype the barcode sequence may then be analyzed by sequencing. The phenotype is then linked to a genotype by matching the barcode of the protein of interest having a phenotype of interest with a cDNA having the same barcode.

0019However, as it will be understood by the skilled in the art, such a method does not allow to match a whole transcriptome of a single cell with the phenotype of a single cell or to access the genotypes of single cells having a phenotype which is not associated with the presence of one particular protein of interest.

0020The international patent application <patcit id="pcit0007" dnum="WO2016207441A"><text>WO2016207441</text></patcit> discloses a method for co-localizing a particle comprising DNA and/or RNA with a known barcode oligonucleotide in a microfluidic droplet. The method is carried out by fusing droplets comprising the particle and the known barcode oligonucleotide with a second droplet species comprising reverse transcriptase mix before incubation to initiate reverse transcription of cellular mRNAs.

0021Contrary to this the method of the inventors allows to phenotype, in one step, several single cells, and then to genotype, in parallel, those single cells by pooling their nucleic acids. Afterwards the genomic information is linked to the phenotype of a single cell having a phenotype of interest.

0022Furthermore, the process of the inventors allows determining any phenotype including a phenotype that is not necessarily associated with the presence of a single protein of interest but which might be associated with a change of activity of, for example certain pathways, and which results in any phenotype that might be determined using an assay, for example, antibody secretion rate, ion channel activity, GPCR activations, a change the Redox potential of a cell.

Detailed description of the invention

Definitions

0023Unless otherwise defined, scientific and technical terms used in connection with the disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art.

0024Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

0025Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art.

0026By "<u>microfluidic</u>", it is generally meant that the dimensions of the passages in which the fluid circulates are smaller than one millimeter and are comprised, for example, from 1 µm and 1 mm.

0027"<u>Microfluidic processes</u>" generally refer to processes wherein small amounts of fluids (10^-4 to 10^-18 liters) are manipulated using microfluidic channels with dimension smaller than one millimeter. Those microfluidic channels are usually contained within a microfluidic device, more particularly within the microfluidic chip of a microfluidic device. Microfluidic devices that are used in context of the present invention are further described herein below in the section "Microfluidic devices".

0028The "<u>process</u>" of the invention may also be referred to as a method.

0029Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The practice of the disclosure will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., <nplcit id="ncit0008" npl-type="b"><text>Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989</text></nplcit>); <nplcit id="ncit0009" npl-type="b"><text>Maniatis et al. Molecular Cloning: A Laboratory Manual (1982</text></nplcit>); <nplcit id="ncit0010" npl-type="b"><text>DNA Cloning: A Practical Approach, vol. I &amp; II (D. Glover, ed</text></nplcit>.); <nplcit id="ncit0011" npl-type="b"><text>Oligonucleotide Synthesis (N. Gait, ed., 1984</text></nplcit>); <nplcit id="ncit0012" npl-type="b"><text>Nucleic Acid Hybridization (B. Hames &amp; S. Higgins, eds., 1985</text></nplcit>); <nplcit id="ncit0013" npl-type="b"><text>Transcription and Translation (B. Hames &amp; S. Higgins, eds., 1984</text></nplcit>); <nplcit id="ncit0014" npl-type="b"><text>Animal Cell Culture (R. Freshney, ed., 1986</text></nplcit>); <nplcit id="ncit0015" npl-type="s"><text>Perbal, A Practical Guide to Molecular Cloning (1984</text></nplcit>).

0030The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

0031The term "<u>nucleic acid</u>" as herein used generally refers to at least one molecule or strand of DNA, RNA, miRNA or a derivative or mimic thereof, comprising at least one nucleobase, such as, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine "A," guanine "G," thymine "T," and cytosine "C") or RNA (e.g. A, G, uracil "U," and C). The term "<u>nucleic acid</u>" encompasses the term "<u>oligonucleotide".</u>

0032"<u>RNA</u>" herein refers to, but is not limited to, functional RNA, such as mRNA, tRNA, rRNA, catalytic RNA, siRNA, miRNA and antisense RNA. In one preferred embodiment, RNA refers to mRNA.

0033As it will be understood by those skilled in the art, the depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. The term nucleic acid thus encompasses complementary DNA. As it will also be appreciated by those skilled in the art, many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. As it will also be understood by those skilled in the art, a single strand nucleic acid, such as, a primer, may hybridize to the target sequence under hybridization conditions, preferably stringent hybridization conditions. Thus, a nucleic acid also encompasses a primer that hybridizes under hybridization conditions to a target sequence.

0034The term "<u>oligonucleotide</u>" refers to at least one molecule of about 3 to about 500 nucleobases in length. For example, the oligonucleotide may have a length of at least 3 nucleobases, at least 10 nucleobases, at least 30 nucleobases, at least 50 nucleobases, at least 100 nucleobases, at least 300 nucleobases, or at least 400 nucleobases. In some cases, the oligonucleotide may have a length of no more than 500 nucleobases, no more than 300 nucleobases, no more than 100 nucleobases, no more than 50 nucleobases, etc. Combinations of any of these are also possible, e.g., the length of the oligonucleotide may be between 3 and 300 nucleobases, preferably 3 and 200 nucleobases, more preferably 3 to 100 nucleobases.

0035These definitions refer to at least one single-stranded molecule, but in some embodiments encompass also at least one additional strand that is partially, substantially or fully complementary to the at least one single-stranded molecule. Accordingly, in some embodiments said definitions refer to double stranded molecules.

0036Thus, in some embodiments, a nucleic acid refers to at least one double-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence comprising a strand of the molecule.

0037"<u>Gene</u>" as used herein may be a genomic gene comprising transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (e.g., introns, 5'- and 3'-untranslated sequences). The coding region of a gene may be a nucleotide sequence coding for an amino acid sequence or a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA and antisense RNA. A gene may also be an mRNA or cDNA corresponding to the coding regions (e.g., exons and miRNA) optionally comprising 5'- or 3'-untranslated sequences linked thereto. A gene may also be an amplified nucleic acid molecule produced in vitro comprising all or a part of the coding region and/or 5'- or 3'-untranslated sequences linked thereto.

0038The term "<u>stringent condition</u>" or "<u>high stringency condition</u>" as used herein corresponds to conditions that are suitable to produce binding pairs between nucleic acids having a determined level of complementarity, while being unsuitable to the formation of binding pairs between nucleic acids displaying a complementarity inferior to said determined level. Stringent conditions are the combination of both hybridization and wash conditions and are sequence dependent. These conditions may be modified according to methods known from those skilled in the art (<nplcit id="ncit0016" npl-type="b"><text>Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York</text></nplcit>). Generally, high stringency conditions are selected to be about 5°C lower than the thermal melting point (Tm), preferably at a temperature close to the Tm of perfectly base-paired duplexes (<nplcit id="ncit0017" npl-type="b"><text>Andersen, Nucleic acid Hybridization, Springer, 1999, p. 54</text></nplcit>). Hybridization procedures are well known in the art and are described for example in <nplcit id="ncit0018" npl-type="b"><text>Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D.,Seidman, J.G., Smith, J. A., Struhl, K. eds. (1998) Current protocols in molecular biology. V.B. Chanda, series ed. New York: John Wiley &amp; Sons</text></nplcit>.

0039High stringency conditions typically involve hybridizing at about 50°C to about 68°C, wherein said temperature typically corresponds to the highest melting temperature TM of the nucleic acid to be hybridized with a target sequence, in 5x SSC/5x Denhardt's solution/1.0% SDS, and washing in 0.2x SSC/0.1% SDS at about 60°C to about 68°C.

0040For instance, in context with the present invention the primer sequence comprised in the at least one oligonucleotide typically hybridizes with a complementary nucleic acid, for example a complementary RNA sequence, at about 50°C to about 68°C in, typically, a reservoir, or a droplet or a plurality of droplets, such as the so-called fused droplet or the plurality of fused droplets. Accordingly, in one example, said fused droplet or said reservoir further comprises a reverse transcriptase composition and, optionally, a lysis composition, wherein the reverse transcriptase composition is defined herein below in the section <i>"reverse transcription"</i> and the lysis composition is defined herein below in the section <i>"cell lysis".</i>

0041The term "<u>antibody</u>" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants of antibodies, including derivatives such as humanized antibodies. In conventional antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non hypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. Complementarity determining regions (CDRs) refer to amino acid sequences which, together, define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding-site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Therefore, an antigen-binding site includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs, i.e. to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species, as defined by <nplcit id="ncit0019" npl-type="b"><text>Kabat, et al. (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1991</text></nplcit>).

0042An "<u>antibody fragment</u>" comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see <patcit id="pcit0008" dnum="US5641870A"><text>U.S. Pat. No. 5,641,870</text></patcit>; <nplcit id="ncit0020" npl-type="s"><text>Zapata et al., Protein Eng. 8(10): 1057-1062 [1995</text></nplcit>]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

0043The term antibody therefore further denotes single chain antibodies, for instance Camelidae antibodies, or nanobodies or VHH.

0044The term "<u>T-cell receptor</u>" herein refers to an antigen-recognition molecule present on the surface of T cells (i.e., T lymphocytes). This definition expressly includes the understanding of the term as known in the art, and includes, for example, a receptor that comprises or consists of a disulfide-linked heterodimer of the highly variable alpha or beta chains expressed at the cell membrane as a complex with the invariant CD3 chains, or a receptor that comprises or consists of variable gamma and delta chains expressed at the cell membrane as a complex with CD3 on a subset of T-cells.

0045"Antibody genes" and "T-cell receptor genes" undergo a unique mechanism of genetic recombination, called V(D)J recombination, that occurs only in developing lymphocytes during the early stages of T and B cell maturation. It involves somatic recombination, and results in the highly diverse repertoire of antibodies/immunoglobulins (Igs) and T cell receptors (TCRs) found on B cells and T cells, respectively.

0046The term "<u>plurality</u>" herein refers to any number which is more than 1 of the object concerned, such as more than 500, more than 1000, more than 2000, more than 5000, more than 100000, such as 1 to 100000, for example 1 to 10000, 1 to 10000, 10 to 1000, 50 to 1000, 100 to 1000, 10 to 500 of the object concerned.

Microfluidic devices

0047The microfluidic processes of the present inventions are performed in microfluidic devices comprising a chip.

0048"<u>Microfluidic chips</u>" generally refer to a set of micro-channels made by milling, etching, ablation or molding into a material (glass, silicon or polymer such as PDMS, PMMA or COC). A microfluidic chip usually comprises a substrate and a support, defining together at least one channel.

0049In some embodiments of the present invention, the microfluidic device used in context of the present inventions comprises a chip comprising a plurality of reservoirs.

0050In some embodiments of the present invention, the microfluidic device used in context of the present inventions comprises a chip comprising at least one microfluidic channel and a plurality of reservoirs.

0051In related embodiments, the at least one microfluidic channel extends in a longitudinal direction (X) between an inlet and an outlet.

0052In related embodiments, each reservoir of the plurality of reservoirs extends along an elevation direction (Z) forming a non-zero angle with the longitudinal direction (X), and each reservoir opens into the at least one channel extending in a longitudinal direction (X).

0053Accordingly, in some embodiments, the microfluidic device comprises a chip comprising: <ul id="ul0001" list-style="dash" compact="compact"><li>at least one microfluidic channel, extending in a longitudinal direction (X) between an inlet and an outlet,</li><li>a plurality of reservoirs, wherein each reservoir is extending along an elevation direction (Z) forming a non-zero angle with the longitudinal direction (X), and each reservoir opening into the at least one channel.</li></ul>

0054In some embodiments, "<u>at least one microfluidic channel</u>" herein refers to at least 1 to 100, 1 to 80, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, such as 1 to 6, 1 to 4, 1 to 3 channels.

0055In some embodiments, the non-zero angle formed between the elevation direction (Z) and the longitudinal direction (X) is from 85 to 95°, preferably 90°.

0056The inlet of the channel is usually used to inject at least one fluid and the outlet of the channel is usually used to collect at least one fluid. The channel is usually intended to allow flowing of the at least one fluid along the main direction from the inlet toward the outlet.

0057The inlet of the channel may be connected with at least one reservoir, such as one to five, preferably one to four, more preferably, one to three, such as one, two or three reservoirs of a fluid intended to flow in the channel.

0058The outlet of the channel may be connected to at least one reservoir, preferably one or two reservoirs, to collect the fluid coming from the channel.

0059In some embodiments, the chip is a drop array, more particularly, a floating drop array.

0060In some embodiments, said microfluidic device further comprises at least one injection device to control the flow rate of the at least one fluid.

0061"<u>At least one fluid</u>" herein may refer to at least one, at least two, at least three, at least four, at least five or more than five fluids.

0062The "<u>at least one fluid</u>" injected at the inlet and the "at least one fluid" collected at the outlet may be the same fluid or different fluids.

0063The injection device may comprise a pressure controller or a syringe driver.

0064In some embodiments, the at least one fluid at the inlet refers to one, two, three or four fluids, for example three fluids.

0065Accordingly, in one example, one fluid contains, for example, droplets of a first type, one second fluid contains, for example, droplets of a second type and a third fluid is to remove a plurality of either the first type or the second type of droplets and to clear out the chamber. The droplets of a first type and a second type are as defined herein below in context of the processes of the invention. According to said example, said microfluidic device further comprises an injection device to control the flow rate of said three fluids.

0066In some embodiments, the device comprises at least one electric system, preferably one electric system, adapted to induce an electric field in a specific area of the chip. The at least one electric system may comprise a generator, an anode and a cathode.

0067In some embodiments, the microfluidic device in context of the invention is used in a device assembly.

0068Said device assembly might further comprise a cell sorter or a unit for producing droplets, such as droplets of the first type or the second type, such as, single cell droplets or RT droplets as further defined herein below.

0069Accordingly, in one alternative embodiment, the chip might comprise a unit for producing droplets of the first type or the second type such as single cell droplets or RT droplets as further defined herein below.

0070Those droplets are then injected via the inlet into the at least one microfluidic channel.

0071"<u>A plurality of reservoirs</u>" herein refers to any number of reservoirs. The number of reservoirs and thus a plurality of reservoirs preferably refers to from 1 to 100000 reservoirs, for example 1 to 10000 reservoirs, such as 10 to 8000, 10 to 7000, 10 to 6000, 10 to 5000, 10 to 4000, 10 to 3000, 10 to 2000, 10 to 1000, for example 1000 to 10000, 1000 to 9000, 2000 to 8000, 3000 to 7000, 4000 to 6000, for example 4500 to 5500.

0072In context of the invention, reference may be made to one reservoir of the microfluidic device. However, the skilled in the art understands that processes described for one reservoir apply in parallel to a plurality of reservoirs present in the chip used.

0073In some embodiment the reservoirs of the chip have the form of a cylinder. In some embodiments, the size of the reservoirs of the plurality of reservoirs is homogenous.

0074It will be understood by the skilled in the art that the microfluidic device, more particularly the chip of the microfluidic device, may be characterized by its geometric parameters, such as, the diameter of one reservoir of the plurality of reservoirs, d<i>_reservoir</i>, depth of the reservoir, h_reservoir, height of the injection channel, h_channel, width of the injection channel, w_channel, and the space between the individual reservoirs of the plurality of reservoirs, x.

Droplets

0075A "<u>droplet"</u> generally refers to a measure of volume. A "<u>droplet</u>" refers in context of the present invention, to an isolated portion of a first fluid that is surrounded by a second fluid. It is to be noted that a droplet is not necessarily spherical, but may assume other shapes as well depending, for example, on the external environment.

0076The term "<u>droplets</u>" used in context of the processes of the invention includes droplets of a first type, droplets of a second type, such as single cell droplets or RT droplets, and fused droplets, or a plurality of said droplets.

0077Methods to prepare and inject droplets are known to the skilled in the art and are further explained herein below in the section "<i>preparation of droplets</i>". With regards to the preparation of droplets, it will be understood by the skilled in the art that the size of the droplets follows a probability distribution, such as a Gaussian distribution. It will be further understood that the parameters used for the preparation of microfluidic droplets can be chosen in order to obtain a plurality of microfluidic droplets having a specific volume.

0078In some embodiments, the droplets in context of the processes of the invention, in particular, the droplets of the first type or the droplets of the second type are preferably, substantially, monodisperse.

0079"<u>Monodisperse</u>" herein refers to droplets having substantially the same shape and/or size.

0080As mentioned herein above, and as known by the skilled in the art, the droplets follow a shape and size distribution.

0081Accordingly, the droplets may have a homogenous distribution of cross-sectional diameters, i.e., the droplets may have a distribution of diameters or volumes such that no more than about 5%, no more than about 2%, or no more than about 1% of the droplets have a diameter or volume less than about 90% (or less than about 95%, or less than about 99%) and/or greater than about 110% (or greater than about 105%, or greater than about 101%) of the overall average diameter or volume of the plurality of droplets. Some techniques for producing homogenous distributions of cross-sectional diameters of droplets are disclosed in International Patent Application No. <patcit id="pcit0009" dnum="US2004010903W" dnum-type="L"><text>PCT/US2004/010903, filed Apr. 9, 2004</text></patcit>, entitled "Formation and Control of Fluidic Species," by Link et al., published as <patcit id="pcit0010" dnum="WO2004091763A"><text>WO 2004/091763 on Oct. 28, 2004</text></patcit>.

0082It will be understood by the skilled in the art, when reference is made herein to a volume or a size of a droplet, this volume or size refers to the average volume or size of the plurality of droplets.

0083Those of ordinary skill in the art will be able to determine the average diameter of a population of droplets, for example, using laser light scattering or other known techniques. The droplets formed in context of the invention can be spherical, or non-spherical in certain cases. The diameter of a droplet, in particular a non-spherical droplet, may be taken as the diameter of a perfect mathematical sphere having the same volume as the non-spherical droplet.

0084The "<u>droplet</u>" or "<u>the plurality of droplets</u>" such as the "<u>plurality of RT droplets</u>" or the "<u>plurality of single cell droplets</u>" have an average volume of less than 5nL, such as less than 4nL, less than 3nL, preferably less than 3nL. In some embodiments, said plurality of microfluidic droplets have an average volume of less than 3nL, less than 2.5nL, less than 2nL, less than 1.5nL, less than 1nL, less than 0.5nL, for example 0.1nL to 3nL, 0.5nL to 3nL, 1nL to 3nL, typically, 10pL, 20pL, 30pL, 50pL, 0.1nL, 0.5nL, 1nL, 1.2nL, 1.4nL, 1.6nL, 1.8nL, 2.0nL, 2.2nL, 2.4nL, 2.6nL, 2.8nL, 3nL.

0085In some embodiments, the droplets of the plurality of microfluidic droplets have an average volume of 1pL to 5000pL, 10pL to 5000pL or 10pL to 3000pL.

0086In some embodiments, the droplets of the plurality of microfluidic droplets have an average volume equal to or less than 1nL.

0087Accordingly, the "<u>fused droplet</u>" or "<u>the plurality of fused droplets</u>" have an average volume of less than 10nL. In some embodiments, said plurality of fused droplets have an average volume of less than 9nL, less than 8nL, less than 7nL, less than 6nL, less than 5nL, less than 4nL, less than 3nL, less than 2nL, less than 1nL, less than 0.5nL, for example 0.1nL to 10nL, 0.1nL to 8nL, 0.1nL to 6nL, 0.1nL to 5nL, such as 0.1nL to 3nL, 0.5nL to 5nL, 0.5nL to 3nL,1nL to 3nL, typically, 0.1nL, 0.5nL, 1nL, 1.2nL, 1.4nL, 1.6nL, 1.8nL, 2.0nL, 2.2nL, 2.4nL, 2.6nL, 2.8nL, 3nL, 4nL or 5nL, such as 11pL to 8000pL.

0088In some embodiments, the plurality of droplets of the first type or the second type, preferably of the first type, have an average diameter that is smaller than the height of the injection channel (h_channel).

0089In some embodiments, the plurality of droplets of the first type or the second type, preferably of the first type, have an average diameter that is 30% to 100% of the height of the injection channel (h_channel), such as 60% to 100%, 70% to 100%; 80% to 100%, 90% to 100%, 92% to 98%, 94% to 98%, for example 95% of the height of the injection channel (h_channel).

0090As it will be understood by the skilled in the art, in particular, the plurality of droplets of the first type have an average diameter that is 30% to 100% of the height of the injection channel (h_channel) as described above, when at least one droplet of the first type among the plurality of droplets moves into one reservoir of said plurality of reservoirs by buoyancy.

0091In some embodiments, the plurality of droplets of the first type or the second type, preferably of the first type, have an average diameter that is 60% to 95%, such as 70% to 90%, 75% to 85%, or 75% to 80% of the diameter of the reservoir (d_reservoir), preferably 75 to 80%.

0092In some embodiments, the plurality of droplets of the second type have an average diameter that is bigger than the height of the injection channel (h_channel), such as an average diameter that is 100% to 250%, 120% to 250%, 150% to 250%, 200 to 250 preferably 200% to 250%.

0093As it will be understood by the skilled in the art, the plurality of droplets of the second type have an average diameter that is bigger than the height of the injection channel (h_channel) as described herein above, when, in the migration step, at least one part of one droplet of the second type enters into one reservoir of said plurality of reservoirs due to the difference in surface energy.

0094In some embodiments, the plurality of droplets of the second type have an average diameter that is smaller or is as large as the droplets of the first type.

0095In some preferred embodiments, the plurality of droplets of the first type or second type, preferably the plurality of droplets of the first type, have an average volume from 1pL to 5000pL, typically 1pL to 4000pL, 1pL to 3000pL, such as 1pL to 500pL, 1pL to 400pL, 1pL to 300pL, 1pL to 200pL or 1pL to 100pL, preferably 1pL to 100pL.

0096In some embodiment, the plurality of droplets of the second type have an average volume that is equal to or an average volume that is higher than the average volume of the plurality of droplets of the first type.

0097In some preferred embodiments, the plurality of droplets of the second type have an average volume from 10pL to 5000pL, 10pL to 4000pL, 10pL to 3000pL, such as 100pL to 4000pL, 100pL to 3000pL, 100pL to 2000pL or 100 to 1000pL, preferably, 100pL to 1000pL.

0098In some embodiments, the plurality of droplets of the second type have an average diameter that is 100% to 250%, such as 150% to 250%, 170% to 230%, 180% to 220%, 190% to 210% of the depth of the reservoir (h_reservoir), for example 200% of the depth of the reservoir (h_reservoir).

0099In some embodiments, the plurality of droplets of the second type have an average diameter that is 70% to 130% of the diameter of the reservoir (d_reservoir), such as 80% to 120%, 90% to 120%, 100% to 120%, 95% to 115%, 95% to 110% of of the diameter of the reservoir, preferably 100 to 120% of the diameter of the reservoir (d_reservoir).

0100In some embodiments, the plurality of droplets of the second type have an average diameter that is bigger than the diameter of the reservoir (d_reservoir), when the droplet of the second type enters partly into a reservoir.

0101In context of the present invention, the microfluidic droplet or plurality of microfluidic droplets comprises an aqueous composition.

0102The <u>"aqueous composition</u>" in context of the invention is, in case of single cell droplets, typically adapted to the cells used and typically comprises a buffered solution as defined herein below. The aqueous composition, in case of RT droplets, may further comprise for example a reverse transcriptase, a reverse transcription composition and/or a lysis composition.

Preparation of the droplets

0103In context of the processes of the invention, in one step, a carrier fluid comprising a plurality of droplets of a first type and, in another step, a carrier fluid comprising a plurality of droplets of a second type are injected into the inlet of the at least one microfluidic channel, preferably one microfluidic channel.

0104The droplets of the first type are either single cell droplets or RT droplets, features of those droplets are further defined herein below and in the sections "<i>microfluidic process for barcoding single cell droplets</i>", "<i>single cell</i>" and "<i>reverse transcription</i>", respectively, wherein the droplets of the second type are RT droplets when the droplets of the first type are single cell droplets or the droplets of the second type are single cell droplets when the droplets of the first type are RT droplets.

0105Those of ordinary skill in the art will be aware of techniques for preparing microfluidic droplets. Techniques for encapsulating cells within microfluidic droplets are described for example in <patcit id="pcit0011" dnum="US7708949B"><text>U.S. Pat. Nos. 7,708,949</text></patcit>, <patcit id="pcit0012" dnum="US8337778B"><text>8,337,778</text></patcit>, <patcit id="pcit0013" dnum="US8765485B"><text>8,765,485</text></patcit>, or Int. Pat. Apl. Pub. Nos. <patcit id="pcit0014" dnum="WO2004091763A"><text>WO 2004/091763</text></patcit> and <patcit id="pcit0015" dnum="WO2006096571A"><text>WO 2006/096571</text></patcit>, <patcit id="pcit0016" dnum="EP2016080341W" dnum-type="L"><text>PCT/EP2016/080341</text></patcit>. Techniques for preparing RT droplets comprising a reverse transcriptase and at least one oligonucleotide can be easily derived by the skilled in the art from the same disclosures, for example <patcit id="pcit0017" dnum="EP2016080341W" dnum-type="L"><text>PCT/EP2016/080341</text></patcit>.

0106In one example, single cell droplets or RT droplets are prepared prior to injection in a separate microfluidic device. In this example, single cell droplets or RT droplets are typically prepared in a microfluidic device having one inlet for a droplet carrier oil (carrier fluid), and additional inlets for components of the droplet aqueous phase. For the carrier oil, typically, fluorinated oil (e.g. HFE-7500) containing, for example, 0.75% (w/w) surfactant (PFPE-PEG-PFPE tri-block copolymer containing two perfluoropolyether blocks (PFPE) and one poly(ethylene)glycol (PEG) block) is typically used. The surfactant is typically used to prevent droplets from coalescing, and the amount may be adjusted, for instance, based on the physicochemical properties of the surfactant used. The carrier oil used for emulsification is not limited to fluorinated liquids and alternative fluids such as fluids based on hydrocarbons (e.g. mineral oil, hexane, etc.), silicon oil and other type of oils can be employed successfully.

0107For single cell droplet preparation one inlet for droplet carrier oil and, usually, one additional inlet is used for delivering a suspension of dissociated cells.

0108For RT droplet preparation, in one example, one inlet for a droplet carrier oil and, typically, two additional inlets are used, delivering (1) a reverse transcriptase and (2) the at least one oligonucleotide.

0109In some embodiments, for RT droplet preparation, for example in context of the processes of the invention, the RT droplets may be fused with other droplets comprising further ingredients, for example, at least one barcode sequence and at least one dye. Methods to fuse flowing droplets are known to the skilled in the art and for example described in <patcit id="pcit0018" dnum="WO2010128157A1"><text>WO2010128157 A1</text></patcit>.

0110As it will be understood by the skilled in the art, for example, in case of single cell droplets, the number of cells encapsulated in one droplet and the size of the droplets follows a probability distribution, for example a Poisson or Gaussian distribution, and depends on concentration of the cells, the geometry of the main channel, the injection parameters of the cell suspension and the carrier fluid.

0111Accordingly, the parameters can be adapted to obtain droplets with either 1 or 0 cells in it, thus avoiding droplets containing several cells.

0112Accordingly, in context of the invention at least some of the single cell droplets comprise one single cell.

0113Cells in context of the present invention comprise nucleic acids, it will be therefore understood by the skilled in the art that the single cell droplets in context of the invention comprising single cells thus comprise single cell nucleic acids.

0114Similar to the preparation of the single cell droplets, it will be understood by the skilled in the art, that in case of RT droplets, the encapsulation of a reverse transcriptase and at least one oligonucleotides follows as well a probability distribution, for example a Poisson or Gaussian distribution, and depends on the concentration of the reverse transcriptase, the concentration of the at least one oligonucleotide, the geometry of the main channel, the injection parameters of the reverse transcriptase, the at least one oligonucleotides and the carrier fluid. Furthermore, in same embodiments, the at least one oligonucleotide comprised in the RT droplets is bound to at least one particle of a first type.

0115Accordingly, the number of particles of a first type encapsulated in one droplet follows as well a probability distribution, for example a Poisson distribution, and depends on concentration of the particles, the geometry of the main channel, the injection parameters of the particle suspension and the carrier fluid.

0116Accordingly, the parameters can be adapted to obtain droplets with 0, 1 or 2 particles in it. Accordingly, in some embodiment, at least some of the RT droplets comprise, preferably, one particle of the first type.

0117The "<u>carrier fluid</u>" is immiscible with aqueous solution of the droplets.

0118"<u>Carrier fluids</u>" used for the preparation of droplets are known to the skilled in the art and are usually fluorinated oils.

0119Accordingly, in some embodiments the carrier fluid is fluorinated oil, such as HFE-7500.

0120Furthermore, in order to prevent droplets from coalescing, the carrier fluid may further comprise a surfactant.

0121Accordingly, in some embodiments, the carrier fluid further comprises a surfactant.

0122Surfactants are usually present at 0.1% to 10%, such as 0.1% to 1% or 1% to 10%, 2%, depending on the physicochemical properties of the surfactant. In one example, the carrier fluid comprises 1.5%, 1% or 0.75% (v/v) surfactant, such as PFPE-PEG-PFPE tri-block copolymer containing two perfluoropolyether blocks (PFPE) and one poly(ethylene)glycol (PEG) block.

0123The surfactant is preferably highly soluble in fluorinated fluids and nearly insoluble in the aqueous phase.

0124However, since the RT and single cell droplets are fused in a later step the choice and concentration of the surfactant used for the preparation of the droplets preferably does not prevent fusion of said droplets.

Single cell

0125As described herein above in the section <i>"preparation of the droplets"</i> encapsulation conditions (conditions to prepare single cell droplets) are preferably chosen in a way that single cell droplets contain 0 or 1 cell. To increase the percentage of droplets containing single cells, the plurality of single cell droplets might be screened and sorted prior to the step of injecting the plurality of single cell droplets.

0126Furthermore, in some embodiments, said plurality of single cell droplets may be screened for a phenotype of interest prior to injecting the plurality of single cells.

0127Accordingly, in some embodiment the single cell droplets are screened and sorted prior to injecting a plurality of single cell droplets.

0128Microfluidic cell sorting techniques are known to the skilled in the art and described, for example, <nplcit id="ncit0021" npl-type="s"><text>Wyatt Schields, C. et al. (Lab Chip. 2015 February 16; 15(5): 1230-1249</text></nplcit>).

0129Examples of microfluidic cell sorting techniques include, but are not limited to, electrophoresis, dielectrophoresis (DEP), electroosmotic flow, acoustophoresis, optical manipulmation, mechanical systems, magnetophoresis, electrokinetic mechanisms, optical tweezers and passive cell sorting. Some methods might also combine several of these techniques.

0130Passive cell sorting herein refers to methods relying on inherent differences and sort cells using inertial forces, hydrodynamic spreading, deterministic lateral displacement, filtration, transient cellular adhesion, and cellular immobilization.

0131In a preferred embodiment, single cell droplets are sorted using acoustophoresis or by dielectrophoresis prior to injecting the plurality of single cell droplets.

0132In one example, preferably, acoustophoresis, in particular, surface acoustic waves are utilized to move selected droplets into different sorting bins, as described for example in <patcit id="pcit0019" dnum="US20130213488A1"><text>US 20130213488 A1</text></patcit>.

0133In a further preferred embodiment, single cell droplets are sorted prior to injecting using the methods described in, for example, <patcit id="pcit0020" dnum="WO2016059182A"><text>WO2016059182</text></patcit>.

0134As known to the skilled in the art, some of these methods (such as magnetophoresis, acoustophoresis or electrokinetic mechanisms) might require labeling of the single cells. However, the type of label and if a label is required might depend on the cells to be analyzed. To give an example, for magnetophoresis, for example, cells might be labeled with a magnetic particle, however, erythrocytes, for example, may be sorted based on their natural iron content. Alternatively, in some examples, some types of cells might be manipulated using ferrofluids or paramagnetic solutions.

0135In most embodiments, the single cell droplets further comprise a fluorescent dye.

0136A "<u>fluorescent dye</u>" herein refers to a fluorescent substance that can re-emit light upon light excitation.

0137In some embodiments, when used, for example, in context of cell sorting, the fluorescent dye is conjugated to an antibody that recognizes a target feature on or in the cell; the dye may also be attached to a chemical entity with affinity for the cell membrane or another cellular structure. In some embodiments, the fluorescent dye recognizes a cellular structure.

0138In some embodiments, a fluorescent dye includes, but is not limited to reactive and conjugated dyes, nucleic acid dyes, cell function dyes, and fluorescent proteins or fluorescent nanoparticles.

0139"<u>Reactive and conjugated dyes</u>" include, but are not limited to xanthene (such as fluorescein), rhodamine, coumarine and cyanine dyes or derivatives thereof such as the Alexa fluor dyes.

0140"<u>Nucleic dyes</u>" include, but are not limited to, Sytox dyes, DRAQ7, propidium iodide (PI), and 7-aminoactinomycin D (7-AAD).

0141"<u>Amine dyes</u>" bind to the amine groups of cellular proteins. Those dyes bind to proteins of the cellular surface or to intracellular proteins of dead cells.

0142"<u>Cell function dyes</u>" are dyes that become fluorescent in specific metabolic conditions of a cell and include, for example, calcein. Calcein becomes fluorescent when binding intracellular Ca²⁺.

0143"<u>Fluorescent proteins</u>" are known to the skilled in the art and include but are not limited to GFP or EBFP.

0144"<u>Fluorescent nanoparticles</u>" include but are not limited to, for example, PAN particles.

0145As it will be understood by the skilled in the art, some of the sorting techniques mentioned above, might require the introduction of a particle into the single cell droplet.

0146Accordingly, in some embodiments, the single cell droplets might further comprise at least one particle of a second type.

0147In some embodiments, the at least one particle of a second type herein refers to at least 1, at least 2 at least 3, such as more than one, such as 1 to 10, 1 to 5, 1 to 3 particles.

0148In some embodiments, the particle of a second type is fluorescent.

0149In some embodiments, said particle of a second type is selected from the group consisting of elastomeric particle, hydrogel particle, a polymeric particle or a magnetic particle, preferably a hydrogel particle or a magnetic particle, wherein the magnetic particle and the hydrogel particle are as defined herein below in the section <i>"oligonucleotides".</i>

0150In some embodiments the magnetic particle is a paramagnetic particle or a super paramagnetic particle.

0151"<u>Elastomeric particles</u>" herein refer for example to silicone elastomeric particles.

0152In some embodiments, the particle of a second type is functionalized so that they could have other molecules attached, such as proteins, nucleic acids or small molecules. Methods to functionalize a particle are known to the skilled in the art.

0153In some embodiments, the particle of a second type is functionalized with antibodies. The particle of a second type may be functionalized with an antibody using a, for example, a streptavidin-biotin link, as described herein below in the section RT-droplets in context with the particles of a first type.

0154In some embodiment, said antibody is directed against a component of the single cell.

0155The "<u>cell</u>" in context of the present invention is given its ordinary meaning as used in biology, for example a cell refers to an autonomous self-replicating unit that may exist as functional independent unit of life, for example for unicellular organism, or as sub-unit in a multicellular organism, for example in plants and mammals, that is specialized into carrying out particular functions towards the cause of the organism as a whole. However a "cell" may further refer to quiescent cells which typically are still capable of cell division when mitotic stimulation is applied.

0156In some embodiments, cells refer to prokaryotic cells or eukaryotic cells, preferably, eukaryotic cells.

0157The defining feature distinguishing an "<u>eukaryotic cell</u>" from a prokaryotic cell is that they have membrane-bound organelles, especially the nucleus, which contains the genetic material, and is enclosed by the nuclear envelope.

0158An "<u>eukaryotic cell</u>" in context of the present invention is selected from the group consisting of a mammal cells, plant cell and fungal cell, preferably mammalian cell.

0159A "<u>mammal</u>" herein refers to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.

0160Accordingly, in some embodiments, the cell is a mammalian cell, an engineered mammalian cell or a cell line or a mammalian immune cell.

0161In some embodiments, the mammalian cell is an immune cell.

0162In some embodiments, an immune cell may be, but is not limited to, B cells, T cells, or hybridomas, preferably B cell.

0163In some embodiments, a cell or plurality of cells herein refers to different types of cells or to cells of the same type or origin exposed to different conditions.

0164In one particular embodiment, the cell is a non-mammalian cell.

0165In one further particular embodiment, the non-mammalian cell is a yeast cell, an avian cell or a shark cell.

0166As mentioned above, the single cell droplets usually comprise an aqueous composition, wherein said aqueous composition is typically adapted to the cells used and typically comprises a buffered solution as defined herein below.

0167As known to the skilled in the art cells contain nucleic acids, wherein nucleic acids are as defined herein above.

0168The cells in context of the processes of the invention are preferably living cells. Methods to separate living from dead cells are known to the skilled in the art.

Cell lysis

0169As known to the skilled ion the art, the nucleic acid present in a single cell are released when said cells are lysed.

0170"<u>Cell lysis</u>" used in context of the present invention may be accomplished by enzymatic, physical, and/or chemical means, or any combination thereof, in particular enzymatic, physical, and/or chemical means. Other cell disruption methods may be also be used. The cell lysis methods described herein below apply to the lysis of the cell when present in a droplet, such as the single cell droplet or fused droplet, or when present in the reservoir without a droplet.

0171Accordingly, in some embodiments, the single cell or cell is lysed in the lysis step in context of the processes of the invention using enzymatic, physical, and/or chemical cell lysis.

0172"<u>Enzymatic methods</u>" to remove cell walls is well-established in the art. The enzymes are generally commercially available and, in most cases, were originally isolated from biological sources. Enzymes commonly used include lysozyme, lysostaphin, zymolase, mutanolysin, glycanases, proteases, and mannose.

0173As known by the skilled in the art "<u>chemical cell lysis</u>" is achieved using chemicals such as detergents, which disrupt the lipid barrier surrounding cells by disrupting lipid-lipid, lipid-protein and protein-protein interactions. The ideal detergent for cell lysis depends on cell type and source. Nonionic and zwitterionic detergents are milder detergents. The Triton X series of nonionic detergents and 3-[(3-Cholamidopropyl)dimethylammonio]-I-propanesulfonate (CHAPS), a zwitterionic detergent, are commonly used for these purposes. In contrast, ionic detergents are strong solubilizing agents and tend to denature proteins, thereby destroying protein activity and function. SDS, an ionic detergent that binds to and denatures proteins, is used extensively in the art to disrupt cells.

0174"<u>Physical cell lysis</u>" refers to the use of sonication, heat shock, ice shock or electroporation.

0175In one example, the cells are lysed using cold shock. This might be achieved in context of the processes of the invention by cooling down, for example, the chip comprising single cells in the plurality of reservoirs.

0176As mentioned herein below, in some embodiments, the RT droplet or plurality of RT droplets, fused droplet or plurality of fused droplets, or the plurality of reservoirs comprise a lysis composition.

0177In some embodiments, the lysis composition comprises enzymes selected from the group consisting of lysozyme, lysostaphin, zymolase, mutanolysin, glycanases, proteases, and mannose.

0178In one preferred embodiment, the lysing composition in context of the present invention comprises magnesium chloride, a detergent, a buffered solution and an RNase inhibitor.

0179In some embodiments, the detergent is selected from the group consisting of Triton-X-100, NP-40, Nonidet P40, and Tween-20 and IGEPAL CA 630.

0180Nonlimiting examples of the buffered solution may include Tris-HCI, Hepes-KOH, Pipes-NaOH, maleic acid, phosphoric acid, citric acid, malic acid, formic acid, lactic acid, succinic acid, acetic acid, pivalic (trimethylacetic) acid, pyridine, piperazine, picolinic acid, L-histidine, MES, Bis-tris, bis-tris propane, ADA, ACES, MOPSO, PIPES, imidazole, MOPS, BES, TES, HEPES, DIPSO, TAPSO, TEA (triethanolamine), N-Ethylmorpholine, POPSO, EPPS, HEPPS, HEPPSO, Tris, tricine, Glycylglycine, bicine, TAPS, morpholine, N-Methyldiethanolamine, AMPD (2-amino-2-methyl-1,3-propanediol), Diethanolamine, AMPSO, boric acid, CHES, glycine, CAPSO, ethanolamine, AMP (2-amino-2-methyl-1-propanol), piperazine, CAPS, 1, 3-Diaminopropane, CABS, or piperidine (see also, www.reachdevices.com/Protein/BiologicalBuffers.html). Nonlimiting examples of RNase inhibitors may include RNase OUT, IN, SuperIN Rnase, and those inhibitors targeting a wide range of RNAse (e.g., A, B, C, 1 and T1).

0181As mentioned herein below in the section "<i>Microfluidic process for barcoding single cell nucleic acids</i>", the chemical lysis, when applied to droplets, occurs, typically after fusing the single cell droplet with the RT droplet. In some embodiments, the cell lysis occurs preferably after performing a phenotypic assay, as for example after step b) of the process for genotyping single cells having a phenotype of interest.

0182As it will be understood by the skilled in the art, the components such as the reverse transcriptase and reverse transcriptase composition and the lysis composition will be diluted when further components are added, for example, the lysis composition will be diluted when, in one example, the RT and single cell droplets are fused.

0183Accordingly, the concentrations given for the ingredients of the lysis composition are preferably given as final concentrations, for example after droplet fusion. The skilled in the art will thus understand to adapt the initial concentrations present in the reservoir, or the RT droplets.

0184In some embodiments, the concentration of magnesium chloride is 1 mM to 20 mM.

0185In some embodiments, the concentration of the detergent is 0.1% to 10%.

0186In one example, the lysis composition is typically 0.2% Triton, 3mM MgCl₂, 50mM Tris-HCI pH 7.4.

Reverse transcription (RT)

0187In one step in context of the processes of the invention a plurality of RT droplets is injected, wherein at least some of the RT droplets comprise a reverse transcriptase (RT) and at least one oligonucleotide.

0188In context of the microfluidic process for genotyping single cells having a phenotype of interest the reverse transcriptase (RT) and at least one oligonucleotide is provided in each reservoir of the plurality of reservoirs.

0189A "<u>reverse transcriptase (RT)</u>" is an enzyme used to generate complementary DNA (cDNA) from a nucleic acid template, in particular a RNA template, in a process termed reverse transcription.

0190In some embodiments, the reverse transcriptase is selected from the group consisting of Superscriptase I, Superscriptase II, Superscriptase III, Superscriptase IV, Murine Leukemia RT, SmartScribe RT or MultiScribe RT.

0191In some embodiments, the reverse transcriptase has a template switch activity.

0192The template switch activity permits to uniquely label cDNA with a universal sequence. Using the template switch activity leads to uniquely labelling cDNA at its 5'end with said universal sequence. A universal sequence herein refers to sequences typically used for 5' Rapid Amplification of cDNA End (RACE). It is generally known to the skilled in the art how to perform 5'RACE and which universal sequence may be used.

0193In context of the processes of the present invention, reverse transcription takes place either after fusing the at least one droplet of the first type with at least one droplet of the second type or, optionally, when the process is performed without droplets, after step c) of the process of genotyping single cells having a phenotype of interest.

0194Accordingly, as described herein above in context of the lysis composition, it will be understood by the skilled in the art, that ingredients, such as the reverse transcriptase will be diluted when the different ingredients such as a lysis composition, the reverse transcriptase or the reverse transcription composition are mixed. Accordingly, the concentrations given for any ingredients herein below are preferably given as final concentrations, present, for example, after droplet fusion.

0195In some embodiments, the concentration of the reverse transcriptase is 1 to 50U/µl, preferably 5 to 25U/µl, for example at 12.5U/µl.

0196"<u>Reverse Transcription</u>" or "<u>RT reaction</u>" is a process in which single-stranded RNA is reverse transcribed into a single-stranded complementary DNA (cDNA) by using total cellular RNA or poly(A) RNA, a reverse transcriptase enzyme, a primer, dNTPs and an RNase inhibitor. It will be understood by the skilled in the art, that the product of the reverse transcription is a RNA/DNA duplex comprising a single strand cDNA hybridized to its template RNA. As it will be further understood, said RNA/DNA duplex is further linked to the at least one oligonucleotide comprising the primer sequence used for the reverse transcription.

0197Accordingly, it will be understood by the skilled in the art that reverse transcribing the nucleic acids, for example in step b) of the microfluidic process for barcoding single cell nucleic acids, results in single cell cDNA.

0198Accordingly, in some embodiments, the at least some of the fused droplets further comprise single cell cDNAs produced by reverse transcription of nucleic acids from the single cell lysate.

0199Accordingly, in some embodiments, when no droplets are used, the reservoir further comprises single cell cDNAs produced by reverse transcription of nucleic acids from the single cell lysate.

0200In some embodiments, said cDNA refers to a single-stranded complementary DNA.

0201In a further embodiment, said cDNA is comprised in a RNA/DNA duplex.

0202In some embodiments, the RNA/DNA duplex refers to the RNA that has been reverse transcribed and is still hybridized either partially or over the entire length to the synthesized cDNA and/or the primer sequence of the at least one oligonucleotide.

0203As it will be understood by the skilled in the art, in some embodiments, the RNA/DNA duplex is linked to the at least one oligonucleotide comprising the primer sequence to which the nucleic acid, preferably mRNA was hybridized in the hybridization step, for example in step a), and which was used for reverse transcription in the reverse transcription step, for example in step b).

0204As it will be understood by the skilled in the art, in some embodiments the at least one oligonucleotide and thus the RNA/DNA duplex is linked to a particle of a first type.

0205In some embodiments, the plurality of reservoirs, the RT droplet or plurality of RT droplets or the fused droplet or plurality of fused droplets comprise a reverse transcriptase composition.

0206In some embodiments, a reverse transcriptase composition comprises a protease inhibitor, dNTPs and/or DTT, preferably protease inhibitor, dNTPs and DTT.

0207In some embodiments, the protease inhibitor comprises a plurality of protease inhibitors.

0208In some embodiments, the protease inhibitor is selected from the list consisting of Leupeptin hemisulfate salt, pepstatin A, AEBSF, Aprotinin, Bestatin hydrochloride, E-64 and PMSF.

0209For example, the protease inhibitor may comprise one or more of Leupeptin hemisulfate salt, pepstatin A, AEBSF, Aprotinin, Bestatin hydrochloride, E-64 and PMSF.

0210As used herein, the term "dNTP" refers to a deoxynucleoside triphosphate, e.g. deoxyadenosine-5'-triphosphate (dATP, "A"), deoxycytidine-5'-triphosphate (dCTP, "C"), deoxyguanosine- 5'-triphosphate (dGTP, "G"), deoxythymidine-5'-triphosphate (dTTP, "T") or deoxyuridine-5'-triphosphate (dUTP, "U"). The term "dNTP" is intended to refer also to deoxynucleoside triphosphates comprising modified bases and base analogues that are capable of mimicking the base pairing of A, C, G, T, or U, or that are capable of base pairing in a degenerate mode, e.g., a base that pairs with A or G, C or T, A or C, G or T, G or C, or A or T, called nucleotide analogues. Said nucleotide analogues may be used, for example, for purification, as further explained herein below.

0211The skilled in the art will understand that the concentrations given for the ingredients of the reverse transcriptase composition are preferably given as final concentrations, present, for example, in the fused droplet after droplet fusion.

0212In some embodiments, the concentration of DTT is 1mM to 10mM, preferably 5mM.

0213In some embodiments, the concentration of dNTP is 0.01mM to 10mM, preferably 0.1 to 1mM, more preferably 0.5mM.

0214In some embodiments, a reverse transcriptase composition further comprises a RNase inhibitor. The RNase inhibitor is defined herein above in the section "<i>Cell lysis</i>".

Oligonucleotide

0215The term "<u>oligonucleotide</u>" is as defined herein above.

0216The "<u>at least one oligonucleotide</u>" in context of the microfluidic processes of the invention comprises a primer sequence.

0217A "<u>primer sequence</u>" is typically a short single-stranded nucleic acid, of between 10 to 50 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be captured and then amplified by typically PCR or reverse transcribed by typically RT. The primer sequences are "<u>specific</u>" to the nucleic acids they hybridize to, i.e. they preferably hybridize under stringency hybridization conditions, more preferably under high stringency hybridization conditions, or are complementary to or almost complementary to the nucleic acids they hybridize to, also called target sequence.

0218"<u>Stringent condition</u>" or "<u>high stringency condition</u>" are as defined herein above.

0219Typically, the primer sequence serves as a starting point for nucleic acid synthesis, allowing polymerase enzymes such as nucleic acid polymerase to extend the primer sequence and replicate the complementary strand. A primer sequence may be complementary to and hybridize to a target nucleic acid. In some embodiments, a primer sequence is a synthetic primer sequence. In some embodiments, a primer sequence is a non-naturally-occurring primer sequence. A primer sequence typically has a length of 10 to 50 nucleotides. For example, a primer sequence may have a length of 10 to 40, 10 to 30, 10 to 20, 25 to 50, 15 to 40, 15 to 30, 20 to 50, 20 to 40, or 20 to 30 nucleotides. In some embodiments, a primer sequence has a length of 18 to 24 nucleotides.

0220In some embodiments, the primer sequence is selected from the group consisting of a poly-T sequence, a random DNA sequence, and a gene-specific sequence.

0221A "<u>poly T sequence</u>" as herein referred to is a sequence comprising 10 to 50, 10 to 40, 10 to 30, 10 to 20, 25 to 50, 15 to 40, 15 to 30, 20 to 50, 20 to 40, or 20 to 30 thymine "T". The Poly T sequence hybridizes with the poly A tail present in mRNAs.

0222In some embodiments, the random DNA sequence can be of any suitable length, such as 6 to 50, 6 to 50, 6 to 40, 6 to 30, 6 to 20, 10 to 50, 10 to 40, 25 to 50, 15 to 40, 15 to 30, 20 to 50, 20 to 40, or 20 to 30 nucleotides.

0223In one particular embodiment, the primer sequence is a gene-specific sequence and the gene is selected from the group consisting of antibody heavy chain variable gene, antibody heavy chain constant gene, antibody light chain variable gene, antibody light chain constant gene, alpha T-cell receptor gene (either variable or constant), beta T-cell receptor gene (either variable or constant), delta T-cell receptor gene (either variable or constant) or genes selected for a panel for transcriptome analysis.

0224The term "<u>antibody</u>" in the wording "antibody heavy chain variable gene", "antibody heavy chain constant gene", "antibody light chain variable gene" and "antibody light chain constant gene" is as defined herein above.

0225The term "<u>T-cell receptor</u>" in the wording "alpha T-cell receptor gene", "beta T-cell receptor gene" and "delta T-cell receptor gene" or "gamma T-cell receptor gene" is as defined herein above.

0226The word "<u>gene"</u> is as defined herein above.

0227The skilled in the art will understand that the number of the at least one oligonucleotide present in each reservoir of the plurality of reservoirs or present in one RT droplet will be adapted to the number of different nucleic acids, in particular to the number of RNAs, that are to be transcribed and barcoded from one single cell.

0228Accordingly, "<u>at least one"</u> in the wording "<u>at least one oligonucleotide</u>" refers to the number of different oligonucleotides present in one droplet or reservoir, wherein one oligonucleotide of the at least one oligonucleotide differs from another oligonucleotide by its primer sequence.

0229In some embodiments, "<u>at least one"</u> in the wording "at least one oligonucleotide" refers to at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 at least 10 oligonucleotides. In some embodiments, at least one oligonucleotide refers to 1 to 100 oligonucleotides, 1 to 80, 1 to 60, 1 to 40, 1 to 30, 1 to 20, 1 to 10, preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9 oligonucleotides.

0230It will be understood that "at least one oligonucleotide" thus refers to at least one oligonucleotide which is present in the RT droplet or in each reservoir at a certain concentration and, accordingly, one oligonucleotide from the at least one oligonucleotide thus refers to a plurality of oligonucleotides of the same sequence.

0231It will be further understood by the skilled in the art that when, for example, the transcriptome of a cell is to be transcribed and barcoded, oligonucleotides having a primer sequence specific to all mRNAs will be used, such as poly T primer sequences, as further defined herein below, whereas, when a gene specific transcriptome is to be transcribed and barcoded, oligonucleotides comprising a gene specific primer sequence are used, as further defined herein below.

0232It will be thus understood, that the number of oligonucleotides corresponds to at least the number of specific genes of which the transcriptome is to be transcribed and barcoded.

0233In one specific embodiment, the at least one oligonucleotide is at least two oligonucleotides, wherein one of the at least two oligonucleotides comprises a primer sequence specific to one gene, such as an antibody heavy chain variable gene and wherein the other oligonucleotide comprises a primer sequence specific for another gene, such as an antibody light chain variable gene.

0234In another particular embodiment, the at least one oligonucleotide is at least two oligonucleotides, wherein one of the at least two oligonucleotides comprises a primer sequence specific for, for example, the alpha T-cell receptor gene or the beta T-cell receptor gene or gamma T-cell receptor, and wherein the other of the at least two oligonucleotides comprises a primer sequence specific for, for example, the delta T-cell receptor gene.

0235In another particular embodiment, the at least one oligonucleotide is at least three oligonucleotides, wherein the first of the at least three oligonucleotides comprises a primer sequence specific for, for example, the alpha T-cell receptor gene and the second of the at least three oligonucleotides comprises a primer sequence specific for, for example, the beta T-cell receptor gene, and wherein the third of the at least three oligonucleotides comprises a primer sequence specific for, for example, the polyA mRNA.

0236The "<u>transcriptome</u>" generally refers to the set of all messenger RNA molecules in one cell or a population of cells. Accordingly, the "<u>transcriptome of a cell"</u> or "<u>the transcriptome of a single cell"</u> herein refers to the set of all messenger RNA molecules in one cell.

0237As it will be understood by the skilled in the art, a gene specific transcriptome thus refers to the set of all messenger RNA molecules derived from one gene.

0238As it is known by the skilled in the art, different gene products, so called isoforms, may be encoded by one gene. Accordingly, a gene specific transcriptome may further refer to the messenger RNA molecules of at least one specific isoform of one specific gene, such as the messenger RNA molecules of 1, 2, 3, 4 or more specific isoforms of one specific gene or to the messenger RNA molecules of all isoforms of one specific gene.

0239Accordingly, in some embodiments, the single cell nucleic acid in context of the present invention is RNA, wherein RNA is as defined herein above.

0240In a further embodiment, mRNA comprises a poly A sequence, also called poly A tail.

0241Furthermore, in some particular embodiments, the "<u>at least some of the nucleic</u> acids" in context of the present invention refers to at least one nucleic acid, preferably, at least 2, at least 3, at least 4, at least 5, nucleic acids or more. In one particular, the nucleic acids of step a) and b) refer to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleic acids.

0242The processes of the invention can be used to reverse transcribe from one specific nucleic acid up to all nucleic acids of a single cell.

0243Accordingly, in further embodiments, the "<u>at least some of the nucleic acids</u>" in context of the present invention refers to from 1 to 100000 nucleic acids, such as 1 to 80000, 1 to 60000, 1 to 40000, such as 1, 1000, 2000, 4000, 6000, 8000, 10000, 12000, 14000, 16000, 20000, 25000, 30000, 45000, 50000 nucleic acids. Accordingly, in some embodiments, the nucleic acid herein refers to the RNA of all genes.

0244As mentioned above, the processes of the invention also refer retrotranscribing a gene specific transcriptome.

0245Accordingly, in some embodiments, the nucleic acid herein refers to the RNA of at least one specific gene.

0246Said at least one specific gene may be at least one, at least 2, at least three, at least four at least five, such as 1 to 10, 1 to 5 genes, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 genes.

0247In some embodiments, said at least one specific gene is selected from the group consisting of antibody heavy chain variable gene, antibody heavy chain constant gene, antibody light chain variable gene, antibody light chain constant gene, alpha T-cell receptor gene, beta T-cell receptor gene, and delta T-cell receptor gene and gamma T-cell receptor gene.

0248In some embodiments, specific genes can also be selected to further gain information of cell phenotype and to classify the cells based on gene expression.

0249In some embodiments, the at least one oligonucleotide further comprises a promoter and/or a spacer sequence.

0250The promoter and/or the spacer sequence are preferably located towards the 5' end or located at the 5' end of the primer sequence. Examples of promoter sequences include, but are not limited to, T7 promoters, T3 promoters, or SP6 promoters.

0251In some embodiments, the oligonucleotide further comprises a barcode sequence.

0252Accordingly, in some embodiments the oligonucleotide comprises from 5' to 3' a barcode sequence and a primer sequence.

0253The "<u>barcode sequence"</u> or simply called "barcode" herein refers to a unique nucleic acid sequence that can be distinguished by its sequence from another nucleic acid sequence, thus permitting to uniquely label a nucleic acid sequence so that it can be distinguished from another nucleic acid carrying another barcode sequence.

0254In some embodiments, the barcode sequence uniquely identifies the nucleic acids released by a single cell from nucleic acids released from other cells, for instance, even after the nucleic acids are pooled together.

0255In some embodiments, the barcode sequence is used to distinguish tens, hundreds, or even thousands of nucleic acids, e.g., arising from different cells or other sources.

0256In some embodiments, the barcode sequence is of any suitable length. The barcode sequence is preferably of a length sufficient to distinguish the barcode sequence from other barcode sequences. In some embodiments, a barcode sequence has a length of 1 to 5, 1 to 10, 5 to 15, or more than 15 nucleotides such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 72, 74, 76, 78, 80, 85, 90 or more nucleotides, such as 50 to 85, 60 to 80, 70 to 80 nucleotides.

0257In some embodiments, the barcode sequence consists of more than one barcode sequence and thus may be referred to as "at least one barcode sequence".

0258In a related embodiment, the different barcode sequences may be taken from a "pool" of potential barcode sequences, which themselves have typically been generated by split and poll synthesis. If the barcode sequence consists of more than one barcode sequence, the barcode sequences may be taken from the same, or different pools of potential barcode sequences. The pool of sequences may be selected using any suitable technique, e.g., randomly, or such that the sequences allow for error detection and/or correction, for example, by being separated by a certain distance (e.g., Hamming distance) such that errors in reading of the barcode sequence can be detected, and in some cases, corrected. The pool may have any number of potential barcode sequences, e.g., at least 100, at least 300, at least 500, at least 1,000, at least 3,000, at least 5,000, at least 10,000, at least 30,000, at least 50,000, at least 100,000, at least 300,000, at least 500,000, or at least 1,000,000 barcode sequences.

0259Methods to join different barcode sequences taken from one "pool" or more than one pool are known to the skilled in the art and include, but are not limited to, the use of ligases and/or using annealing or a primer extension method.

0260Non-limiting examples of ligases include DNA ligases such as DNA Ligase I, DNA Ligase II, DNA Ligase III, DNA Ligase IV, T4 DNA ligase, T7 DNA ligase, T3 DNA Ligase, E. coli DNA Ligase, Taq DNA Ligase, or the like. Many such ligases may be purchased commercially.

0261In some embodiments, the barcode sequence is a double stranded or single stranded nucleic acid.

0262In some embodiments the oligonucleotide further comprises a unique molecular identifier (UMI).

0263The UMI is located at the 3' end or the 5' end of the barcode sequence.

0264"<u>Unique molecular identifier"</u> (UMI) sequences are well known to the skilled in the art and are described, for example in <nplcit id="ncit0022" npl-type="s"><text>Kivioja et al (Nature Method, 2012, vol9, N°1</text></nplcit>). The UMI sequences allow bioinformatic removal of duplicated reads.

0265In some embodiments, the at least one oligonucleotide is introduced into the reservoir or the droplets, in particular into the RT droplets, by initially binding the at least one oligonucleotide to at least one particle of a first type.

0266It will be understood by the skilled in the art that binding the at least one oligonucleotides temporally to a particle permits to provide particles having a high amount of oligonucleotides. Furthermore, binding the at least one oligonucleotide initially to the particle facilitates, in particular when droplets are used, the introduction of the at least one oligonucleotides into each droplet, such as the RT droplet.

0267Accordingly, in some embodiments the reservoir or the RT-droplet further comprises at least one particle of a first type to which the at least one oligonucleotide is bound.

0268At least one particle of a first type herein refers to at least one, two, three, four, five or more particles of a first type, preferably one particle of a first type.

0269As it will be understood by the skilled in the art and as explained herein above, the number of the particles of a first type encapsulated in one RT droplet and the size of the droplets follow a probability distribution, for example a Poisson distribution, and depends on the concentration of the particles of a first type, the injection parameters of the aqueous composition, such as the reverse transcription composition or lysis composition, the geometry of the main channel, the injection parameters of the particles of the first type and the carrier fluid.

0270Accordingly, in some embodiments, the parameters can be adapted to obtain RT droplets with 2, 1 or 0 particles of a first type in it, thus avoiding droplets containing more than 2 particles of a first type.

0271In a related embodiment, the parameters can be adapted to obtain RT droplets with 1 or 0 particles of a first type in it, thus avoiding droplets containing more than 1 particle of a first type.

0272In some embodiments, the at least one particle of a first type is encapsulated within the RT droplets at no more than about 2 particles of a first type /droplet, preferably, in a further embodiment, the at least one particle of a first type is encapsulated are encapsulated within the droplets at no more than about 1 particle/droplet, or the at least one particle of a first type is encapsulated within the droplets preferentially with 1 particle/droplet, or the at least one particle of a first type is encapsulated within the droplets with an average of 1 particle/droplet.

0273In analogy, when no droplets are used, in some embodiments, each reservoir preferably comprises no more than about 2 particles of a first type /reservoir, preferably, no more than about 1 particle/reservoir, or 1 particle/reservoir, or an average of 1 particle/reservoir.

0274The "<u>particle"</u>, such as the particle of a first type or particle of a second type, in context of the present invention refers to a microparticle.

0275In some embodiments, the particle is a hydrogel particle, a polymeric particle or a magnetic particle.

0276The particle may have irregular or regular shape. For example, the particle can be spherical, ellipsoidal, or cubic.

0277In some embodiments, the particle in context of the present invention is a hydrogel particle.

0278"<u>Hydrogel particles</u>" are for example described in the International Patent Application No. <patcit id="pcit0021" dnum="WO2008109176A"><text>WO 2008/109176</text></patcit>, entitled "Assay and other reactions involving droplets". Examples of hydrogels include, but are not limited to agarose, poly(ethylene glycol) diacrylate, or acrylamide-based gels, such as bis-acrylamide, polyacrylamide, streptavidine acrylamide, poly-N-isopropylacrylamide, or poly N-isopropylpolyacrylamide or mixtures thereof. In one example the hydrogel particle comprises acrylamide, bis-acrylamide and strepatvidine acrylamide.

0279In another set of embodiments, the particles comprise one or more polymers and are thus herein referred to as "<u>polymeric particle</u>". Exemplary polymers include, but are not limited to, polystyrene (PS), polycaprolactone (PCL), polyisoprene (PIP), poly(lactic acid), polyethylene, polypropylene, polyacrylonitrile, polyimide, polyamide, and/or mixtures and/or co-polymers of these and/or other polymers.

0280In addition, in some embodiments, the particle is magnetic and is thus referred to as "<u>magnetic particle</u>", which could allow for the magnetic manipulation of the particles.

0281For example, the magnetic particles may comprise iron or other magnetic materials.

0282The particles, in particular the particles of a first type or of a second type, could also be functionalized so that they could have other molecules attached, such as proteins, nucleic acids or small molecules. Thus, some embodiments of the present invention are directed to a set of particles defining a library of, for example, nucleic acids, proteins, small molecules, or other species such as those described herein. In one example, said particle is functionalized with an antibody. In some embodiments said antibody is directed against a component of a cell.

0283In some embodiments, the particle, in particular the particle of a first type, is fluorescent.

0284In some embodiments, the particle comprises streptavidin. Streptavidin may be coupled to the surface of the particle defined herein above.

0285In some embodiments the particles of a first or a second type have a size from 0,1pL to 100 pL, such as 0,1pL to 500pL, 0,1pL to 400pL, 0,1pL to 400pL, 0,1pL to 300pL, for example 0,5pL to 300pL, 0,5pL to 250pL, 0,5pL to 200pL, 1pL to 250pL, 1pL to 200pL, preferably 1pL to 200pL., typically 100pL to 200pL, such as 150pL.

0286In some embodiments, the at least one oligonucleotide is covalently linked or non-covalently linked to at least one particle of a first type, wherein the at least one particle of a first type is defined herein above.

0287"<u>Non-covalently linked</u>" herein refers, for example, to a streptavidin -biotin bond. Other non-covalent bonds are known to the skilled in the art, such as avidin biotin bonds or his tag and nickel bonds.

0288"<u>Covalently linked</u>" herein refers for example to an amino bond or an acrylic phosphoramidite bond.

0289"<u>Streptavidin</u>" generally refers to a 52.8 kDa protein purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin with a dissociation constant (Kd) on the order of ≈10^-14 mol/L, the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature.

0290In a preferred embodiment, the non-covalent bond is a streptavidin- biotin link.

0291Streptavidin- Biotin bonds are known to the skilled in the art.

0292Accordingly, in some embodiments, the particle of a first type, for example, comprises streptavidin and in the same embodiment, the at least one oligonucleotide, comprises biotin. In other words, the at least one type of oligonucleotide is functionalized with biotin.

0293Independent of the type of bond used to link the at least one type of oligonucleotide to the particle of a first type, the at least one type of oligonucleotide may further comprise at least one linker sequence.

0294Accordingly, in a further embodiment, the "at least one type of an oligonucleotide" or simply the "oligonucleotide" further comprises at least one linker sequence, said linker sequence is preferably comprised close to the 5' end.

0295In some cases, the oligonucleotide may contain further to the primer sequence mentioned above at one or more primer sequences for sequencing. Examples of such primer sequences for sequencing include, but are not limited to, P5 primer, P7 primer, PE1 primer, PE2 primer, A19 primer, or other primers discussed herein.

0296Accordingly, in some embodiments, the at least one type of an oligonucleotide comprises from 5' to 3' optionally a linker sequence, optionally a promoter sequence, optionally a primer sequence for sequencing, optionally a barcode sequence, optionally a UMI and a primer sequence.

0297It will be thus understood by the skilled in the art that, in some embodiments, the at least one oligonucleotide is bound to the particle of a first type via the 5' end.

0298However, in some alternative embodiments the at least one oligonucleotide may be bound to the particle of a first type via the 3' end.

0299In some embodiments, the "<u>linker sequence</u>" is a sequence with which the at least one oligonucleotide is optionally linked to the particle of a first type.

0300"<u>Optionally linked herein</u>" refers to the possibility that once the at least one type of oligonucleotide linked to the particle is loaded into the RT droplets or the plurality of RT droplets or the reservoirs, the at least one type of oligonucleotide might be released, so that the RT droplet or the fused droplet or the reservoir comprises the particle of a first type and the at least one oligonucleotide without the at least one type of oligonucleotide being linked to said particle of a first type.

0301Preferably, the linker sequence is a cleavable linker sequence, e.g., that can be cleaved upon application of a suitable stimulus, such as enzymatic and/or photocleavage.

0302"<u>Cleavable linkers</u>" may include, but are not limited to, TEV, trypsin, thrombin, cathepsin B, cathespin D, cathepsin K, caspase lumatrix metalloproteinase sequences, phosphodiester, phospholipid, ester, -galactose, dialkyl dialkoxysilane, cyanoethyl group, sulfone, ethylene glycolyl disuccinate, 2-N-acyl nitrobenzenesulfonamide, a-thiophenylester, unsaturated vinyl sulfide, sulfonamide after activation, malondialdehyde (MDA)-indole derivative, levulinoyl ester, hydrazone, acylhydrazone, alkyl thioester, disulfide bridges, azo compounds, 2-Nitrobenzyl derivatives, phenacyl ester, 8-quinolinyl benzenesulfonate, coumarin, phosphotriester, bis-arylhydrazone, bimane bi-thiopropionic acid derivative, paramethoxybenzyl derivative, tert-butylcarbamate analogue, dialkyl or diaryl dialkoxysilane, orthoester, acetal, aconityl, hydrazone, b thiopropionate, phosphoramidate, imine, trityl, vinyl ether, polyketal, alkyl 2-(diphenylphosphino)benzoate derivatives, allyl ester, 8-hydroxyquinoline ester, picolinate ester, vicinal diols, and selenium compounds (see, e.g. Leriche G, Chisholm L, Wagner A.

0303Cleavable linkers are well known to the skilled in the art and are further described in Chemical Biology, for example in <nplcit id="ncit0023" npl-type="s"><text>Leriche H. et al. (Bioorg Med Chern. 15; 20(2):571-82. 2012</text></nplcit>). Cleavage conditions and reagents include, but are not limited to, enzymes, nucleophilic/basic reagents, reducing agents, photo-irradiation, electrophilic/acidic reagents, organometallic and metal reagents, and oxidizing reagents.

0304In some embodiments, the processes of the invention further comprise a step of releasing the at least one oligonucleotide bound to the at least one particle of a first type from said particle, for example, after it has been incorporated into the RT droplets or after it has been provided in the reservoir.

0305Furthermore, in some embodiments, the at least one oligonucleotide initially bound to the at least one particle of a first type is released from said at least one particle of a first type prior to reverse transcribing the single cell nucleic acids.

0306In some embodiments, the step of releasing the at least one oligonucleotides may occur prior or after fusing the single cell and the RT droplets, after lysing the cells and/or before reverse transcribing the single cell nucleic acids nucleic acids.

0307The skilled in the art will understand that depending on the time point selected for releasing the at least some of the oligonucleotides, the term "<u>at least some of the oligonucleotides"</u> might refer to, for example, at least some of the oligonucleotides hybridized to the nucleic acids released by the cells or to a DNA/RNA duplex, as defined herein above.

0308In some embodiments, the at least some of the oligonucleotides can be released using any means, such as enzymes, nucleophilic/basic reagents, reducing agents, photo-irradiation, electrophilic/acidic reagents, organometallic and metal reagents, and oxidizing reagents.

0309In some embodiments, the at least one oligonucleotide can be released using enzymatic and/or photo cleavage. For example, an endonuclease may be used to cleave a linker sequence or any other sequence to release the at least some of the oligonucleotides from the particle of a first type.

0310In a further embodiment, releasing the oligonucleotide refers to disrupting the bond, such as a streptavidin biotin bond. Methods to disrupt a streptavidin biotin bond are known to the skilled in the art and include enzymatic digestion of streptavidin and/or denaturation of streptavidin.

0311In some embodiments, the oligonucleotide is released by enzymatic digestion of streptavidin.

0312The concentration of the at least one oligonucleotide is at least 10nM, preferably at least 100nM.

0313In some embodiments, the concentration of the at least one oligonucleotide is at least 150nM, at least 200nM, at least 300nM, at least 400nM, at least 500nM, at least 600nM, at least 700nM, at least 800nM, at least 900nM and at least 1 µM, such as for instance 100nM to 5µM, 100nM to 4µM, 100nM to 3µM, 100nM to 2µM, 100nM to 1µM, preferably 100nM to 500nM.

0314In one example, the primer sequence is a poly T primer sequence and the concentration of the at least one oligonucleotide is 100nM to 3300nM (corresponding to 3.3µM).

0315In a further example, the primer sequence is a gene specific primer sequence and the concentration of the at least one oligonucleotide is 100nM or 1000nM (corresponding to 1µM).

0316In some embodiments, the concentration of each oligonucleotide of the at least one oligonucleotide in the microfluidic droplets is at least 10nM, preferably at least 100nM.

0317In some embodiments, the concentration of each oligonucleotide of the at least one oligonucleotide is at least 150nM, at least 200nM, at least 300nM, at least 400nM, at least 500nM, at least 600nM, at least 700nM, at least 800nM, at least 900nM and at least 1 µM, such as for instance 100nM to 5µM, 100nM to 4µM, 100nM to 3µM, 100nM to 2µM, 100nM to 1µM, preferably 100nM to 500nM.

0318According to the above, in some embodiments, the reservoir, the RT droplet or plurality of RT droplets further comprise a lysis composition, wherein said lysis composition is defined herein above in the section <i>"cell lysis".</i>

0319In some embodiments, the concentration of the at least one oligonucleotide refers to the concentration in the reservoir, in the RT droplet or in the fused droplet.

0320In some embodiments the RT droplets may be screened and sorted prior to injecting a plurality of RT droplets.

0321Accordingly, in some embodiments the RT droplets further comprise a particle of a second type, wherein said particle of a second type is as defined in the section "single cells".

0322Accordingly, in some further embodiments, the RT droplets further comprise a dye as defined herein above.

Amplifying and Sequencings

0323In some embodiments the processes of the invention further comprise the step of amplifying the barcoded single cell cDNA after recovering said barcoded single cell cDNA. In some embodiments, said amplification step is performed after removing unincorporated oligonucleotides. In some embodiments, said amplification step is performed prior to the sequencing step defined herein below.

0324In some embodiments, the amplifying step is performed in a multiplex reaction, a separated polymerase chain reaction (PCR), isothermal amplification, or a linear amplification.

0325In some embodiments, the linear amplification is an <i>in vitro</i> transcription.

0326In some embodiments, the barcoded single cell cDNA produced in the processes of the invention is quantified using qPCR, such as simplex and/or multiplex qPCR reaction.

0327In a further embodiment the processes of the invention further comprise a step of sequencing the barcoded single cell cDNA.

0328In context of the present invention, in some embodiments, the step of sequencing the barcoded single cell cDNA herein refers to first contacting the barcoded single cell cDNA to a sequencing library and amplifying the sequences of interest from the sequencing library that correspond to the barcoded single cell cDNA, respectively.

0329In some embodiments, the step of sequencing the barcoded single cell cDNA may comprise performing a next generation sequencing (NGS) protocol on a sequencing library.

0330In certain embodiments, the NGS protocol comprises loading an amount of the sequencing library between 4pM to 20pM per flow cell of a reagent kit.

0331In some embodiments, the NGS sequencing protocol further comprises the step of adding 5-60% PhiX to the amount of the sequencing library or to the flow cell of the reagent kit.

Microfluidic process for barcoding single cell nucleic acids

0332"<u>Barcoding</u>" herein refers to adding a genetic sequence, a so-called barcode sequence as defined herein above in the section "<i>oligonucleotide",</i> to a nucleic acid which allows distinguishing said barcoded nucleic acid from a nucleic acid having another added genetic sequence, <i>i.e.</i> another unique barcode sequence.

0333The inventors have developed a process, wherein, in a microfluidic device comprising a chip, one single cell droplet comprising nucleic acids is specifically fused with a droplet comprising a reverse transcriptase and an oligonucleotide having a primer sequence. The nucleic acids are then reverse transcribed and ultimately barcoded during reverse transcription or afterwards. One advantage of this process is that little amounts of material are wasted, since one single cell droplet is specifically fused with one RT droplet. A further advantage is that unused barcodes are removed and that thus the signal to noise ratio is improved when the cDNA is sequenced.

0334A further advantage is that the barcode itself could be used as primer to continue the single cell RT-PCR.

0335Accordingly, the present invention refers to a microfluidic process for barcoding single cell nucleic acids, said method comprises: <ul id="ul0002" list-style="dash" compact="compact"><li>providing a microfluidic device comprising a chip comprising at least one microfluidic channel and a plurality of reservoirs,</li><li>injecting into the inlet of the microfluidic channel a carrier fluid comprising a plurality of droplets of a first type dispersed in the carrier fluid, wherein the droplets of the first type are either single cell droplets or RT droplets, wherein at least some of the RT droplets comprise a reverse transcriptase and at least one oligonucleotide, and wherein at least some of the single cell droplets comprise one single cell, wherein said single cell comprises single cell nucleic acids,</li><li>for a plurality of reservoirs, a first migration step, wherein at least one droplet of the first type among the plurality of droplets is moved into one reservoir of said plurality of reservoirs by buoyancy,</li><li>injecting into the inlet of the microfluidic channel, a carrier fluid comprising a plurality of droplets of a second type dispersed in the carrier fluid, wherein the droplets of the second type are either single cell droplets or RT droplets, and wherein the droplets of the second type are RT droplets when the droplets of the first type are single cell droplets or the droplets of the second type are single cell droplets when the droplets of the first type are RT droplets,</li><li>for a plurality of reservoirs, a second migration step, wherein, at least one part of at least one droplet of the second type enters into one reservoir of said plurality of reservoirs,</li><li>for a plurality of reservoirs, fusing, in or at the edge of each reservoir, said at least one droplet of the first type with said at least one droplet of the second type, thereby resulting in a fused droplet,</li><li>and further comprising the steps of: <ol id="ol0001" compact="compact"><li>a) hybridizing, for each fused droplet, the at least some of the single cell nucleic acids with the at least one oligonucleotide in said fused droplet,</li><li>b) reverse transcribing, in each fused droplet, at least some of the single cell nucleic acids present in said fused droplet, thereby resulting in single cell cDNA, and</li><li>c) attaching at least one barcode sequence to the single cell cDNA obtained in step b), wherein said at least one barcode sequence encodes the identity of said single cell,</li></ol> or</li><li>further comprising the steps of: <ol id="ol0002" compact="compact"><li>a) hybridizing, for each fused droplet, at least some of single cell nucleic acids from at least one cell with the at least one oligonucleotide in said fused droplet, wherein said at least one oligonucleotide comprises at least one barcode sequence,</li><li>b) reverse transcribing, in each fused droplet, at least some of the single cell nucleic acids present in said fused droplet, thereby resulting in barcoded single cell cDNA, wherein said at least one barcode sequence encodes the identity of said single cell.</li></ol></li></ul>

0336Techniques to prepare and inject single cell droplets or RT droplets are well known to the skilled in the art and are further described in the section <i>"preparation of droplets".</i>

0337In some embodiments, the droplets of the first type are single cell droplets. In another set of embodiments, the droplets of the first type are RT droplets. Features relating to the single cell droplets and the RT droplets are described herein above in the previous sections, in particular, in the sections <i>"droplets", "single cell", "reverse transcription"</i> and <i>"oligonucleotides".</i> Accordingly, the description referring to "single cell droplets" or "RT droplets" provided_in these sections are entirely applicable to this section called <i>"microfluidic process for barcoding single cell nucleic acids".</i>

0338The "<u>at least one oligonucleotide"</u> in context of the microfluidic processes of the invention comprises a primer sequence. The at least one oligonucleotide and primer sequence are defined herein above in the section <i>"oligonucleotides".</i>

0339In some embodiments, in the first migration step, the "<u>at least one droplet of the first</u> type" refers to 1, 2 or 3, preferably 1 or 2, more preferably 1 droplet of the first type.

0340Accordingly, in preferred embodiments, in the first migration step, for a plurality of reservoirs, one droplet of the first type is received in each reservoir by buoyancy.

0341"<u>Buoyancy"</u> generally refers to an upward force exerted by a fluid that opposes the weight of an immersed object. In context of the present invention, buoyancy refers to an upward force exerted by the carrier fluid that opposes the weight of a droplet immersed in said carrier fluid.

0342Accordingly, in some embodiments, the microfluidic device is oriented so that the plurality of reservoirs are above the channel and the at least one droplet of the first type enters in the above oriented reservoir by buoyancy. In this embodiment, the carrier fluid has a density that is higher than the density of the aqueous solution.

0343In some embodiments, the first migration step of the at least one droplet of a first type results in an occupancy of 90% to 100% of the plurality of reservoirs, such as 60% to 100%, 70% to 100%, 90% to 100%, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%, preferably the migration step of the at least one droplet results in an occupancy of 90% to 100% of the plurality of reservoirs.

0344"<u>Occupancy</u>" herein refers to the number of reservoirs in comparison to the total number of reservoirs that are occupied with at least one droplet. Occupancy of 50%, for example, means that 50% of the reservoirs are occupied with at least one droplet.

0345In some embodiments, the first migration step of the at least one droplet of a first type results in a capturing efficiency of 10% to 100%, such 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%; 90% to 100% of the injected droplets, preferably 80% to 100%.

0346"<u>Capturing efficiency</u>" refers to the number of droplets in comparison to the total numbers of droplets that were injected into the inlet that are captured in reservoirs. Capturing efficiency of 50%, for example, means that 50% of the total number of the plurality of droplets that were provided are captured in reservoirs.

0347In some embodiments, in the second migration step of the at least one droplet of the second type refers to 1, 2 or 3, preferably 1 or 2, more preferably 1 droplet of the second type.

0348In the second migration step, the at least one droplet of a second type might either migrate completely into the reservoir or only partly, depending on the dimensions of the reservoir, the dimensions of the droplet of the second type and the dimensions of the droplet of the second type. Those dimensions are further defined in the section <i>"droplets"</i> and <i>"microfluidic device".</i>

0349Accordingly, in some embodiments, the at least one part of the at least one droplet of the second type refers to 50% to 100%, such as 60% to 100%, 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, preferably 90% to 100% of the total volume of the at least one droplet of the second type.

0350Accordingly, in an alternative embodiment, the at least one part of the at least one droplet of the second type refers to 1 to 50%, 1% to 40%, 1% to 30%, such 2% to 30%, 5% to 30%, 10% to 30% of the total volume of the at least one droplet of the second type, preferably 10% to 30%.

0351In some embodiments, for a plurality of reservoirs, in the second migration step, at least one part of at least one droplet of a second type enters into one reservoir of said plurality of reservoirs due to buoyancy or the difference in surface energy, preferably the difference in surface energy.

0352In some embodiments, the second migration step of the at least one droplet of a second type results in an occupancy of 50 to 100% of the plurality of reservoirs, such as 60% to 100%, 70% to 100%, 90% to 100%, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%, preferably the migration step of the at least one droplet of a second type results in an occupancy of 90% to 100% of the plurality of reservoirs.

0353In some embodiments, the second migration step of the at least one droplet of a second type results in a capturing efficiency of 10% to 100%, such 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%; 90% to 100% of the injected droplets, preferably 80% to 100%.

0354"Occupancy" and "Capturing efficiency" are as defined herein above.

0355In some embodiments, the droplets of the first type or the droplets of a second type might further comprise at least one particle of a second type, wherein said at least one particle of a second type is defined herein above in the section <i>"single cells"</i> and the particle is defined herein above in the section <i>"oligonucleotides".</i>

0356In some embodiments, the process further comprises a step of, for a plurality of reservoirs, performing for each reservoir a phenotypic assay on said at least one single cell.

0357As it will be understood by the skilled in the art, in some embodiments, performing a phenotypic assay might further require exposing the cell to reagents. Accordingly, in some embodiments, single cells are exposed to one or more reagents before, during, or after parameter measurement.

0358Accordingly, in some embodiments, the single cell droplets further comprise one or more reagents that are required for performing said single cell assay.

0359Accordingly, in some embodiments, the one or more reagents are integrated into the single cell droplet.

0360In some embodiments, the phenotypic assay is performed before the step of fusing the droplets. In some embodiments, the phenotypic assay is performed before lysing the cells. The phenotypic assay might further include the step of taking an image thereby mapping the phenotype of said at least one single cell for each reservoir. The "<u>phenotypic assay</u>" and the step of "<u>taking an image"</u> are as defined herein below in the section <i>"process for genotyping single cells having a phenotype of interest".</i>

0361In context of the process of the invention, the fusing step is performed for a plurality of reservoirs. As it will be understood by the skilled in the art, the fusion might be performed, for example, in selected areas of the chip, in particular if an electrical field is used for fusing the droplets. Accordingly, in some embodiments related to the fusion step, the plurality of reservoirs might refer to some reservoirs of the plurality of reservoirs of the chip.

0362As it will be understood by the skilled in the art, in one example, based on the results of a phenotypic assay, the skilled in the art might select a plurality of reservoirs in which the RT droplets and the single cell droplets will be fused.

0363In some embodiments, in the fusing step, for each reservoir, at least one droplet of the first type is fused with at least a droplet of the second type using temperature, electrical field, demulsifier or ionic force, preferably, electrical field. The methods for fusing droplets are known to the skulled in the art and describe in, for example, <patcit id="pcit0022" dnum="WO2010128157A1"><text>WO 2010128157 A1</text></patcit>.

0364In some embodiments, at least one droplet of the first type is fused with at least one droplet of the second type using electrical field, wherein the electric field is 10kHz and 500V for at least 1 second, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10 seconds, typically 8 seconds.

0365In some embodiments, the fusion step results in a fusion efficiency of 80% to 100% between the droplets of the first type and the droplets of the second type, such as 85% to 100%, 90% to 100%, 92% to 100%; 93% to 100%, 94% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, such as 98%, 99%, 100% between the droplets of the first type and the droplets of the second type, preferably 90% to 100%.

0366<u>"Fusion efficiency between the droplets of the first type and the droplets of the second type</u>" herein refers to number of events wherein the provision of one droplet of a first type and one droplet of a second type result in a fused droplet in comparison to the total number of events wherein one droplet of a first type and a second type are provided together.

0367In some embodiments, the process further comprises, for a plurality of reservoirs, the step of lysing the cell, for example, of the least one single cell droplet or the fused droplet, to release the nucleic acids from the single cell into the single cell droplet or the fused droplet.

0368Accordingly, in some embodiments of the processes of the invention the nucleic acids referred to in the hybridizing step, for example in the hybridizing step a), and in the reverse transcription step, for example the reverse transcription step b), are released nucleic acids.

0369The step of lysing the cells may either occur prior to the step of fusing the at least one droplet of the first type with said at least one droplet of the second type or after the step of fusing said droplets. Methods that may be used to lyse the cells are defined herein above in the section <i>"cell lysis".</i>

0370As it will be understood by the skilled in the art the method selected to lyse the cell may depend on the time point chosen for lysis, <i>i.e.</i> prior or after droplet fusion.

0371For example, enzymatic lysis or chemical lysis is typically used after the fusing step, because the components required for the enzymatic or chemical lysis are typically contained in the RT droplets.

0372In one preferred embodiment, the cell lysis does not destroy the droplets used in context of the invention.

0373Accordingly, in some embodiments, the RT droplets or plurality of RT droplets comprise a lysis composition, wherein the lysis composition is defined herein above in the section <i>"Cell lysis".</i>

0374In some embodiments, the method of the invention further comprises at least one purging step.

0375The "<u>at least one purging step"</u> in context of the invention refers to at least one, at least two, at least three, at least four purging steps.

0376For example, the device may typically be purged before injecting the plurality of droplets of the first type to remove residual air.

0377For example, the device may typically be purged after injecting the plurality of droplets of the first type to remove droplets of the first type that did not migrate into the plurality of reservoirs.

0378For example, the device may typically be purged after injecting the plurality of droplets of the second type to remove droplets of the second type that did not migrate, at least partly, into the plurality of reservoirs.

0379Step a) of the process of the invention refers to hybridizing, for each fused droplet, at least some of the single cell nucleic acids with the at least one oligonucleotide in said fused droplet.

0380"<u>Hybridization</u>" herein refers to a phenomenon in which a primer sequence, for example a primer sequence present in the at least one oligonucleotide, anneals to a complementary nucleic acid sequence of the nucleic acids, accordingly, as known by the skilled in the art, the temperature to be used for hybridization depends on the primer sequence and/or the RT enzyme used.

0381In one example, a hybridization step, for example, the hybridization step a) or b) is performed by incubating the droplets for example for 1 h at 55°C to 60°C.

0382The reverse transcription of at least some of the single cell nucleic acids in step b) is further defined herein above in the section <i>"reverse transcription".</i>

0383In some embodiments, the at least one oligonucleotide comprises a primer sequence and at least one barcode sequence, wherein the "at least one oligonucleotide", the "at least one barcode sequence" and the "primer sequence" are defined herein above in the section <i>"oligonucleotides".</i>

0384In related embodiments, the reverse transcription of, for example step b), thus results in barcoded cDNA. According to the different embodiments described herein above said barcoded cDNA may be bound to a particle of a first type or not.

0385In some embodiments, the process of the invention further comprises the step of recovering the fused droplets at the outlet of the channel after the reverse transcription step.

0386"<u>Recovering</u>" herein refers to collecting the microfluidic droplets comprising cDNA or barcoded cDNA, produced by reverse transcription.

0387In some embodiments, recovering the fused droplets comprises a step of rotating the device around the X-Axis by 45° to 135°, preferably 90° and purging the device. The fused droplets can then be collected at the outlet.

0388In some embodiments, the step of recovering the fused droplets at the outlet of the channel after the reverse transcription step includes at least one washing step.

0389After washing the fused droplets, the downstream processing of the fused droplets may differ for droplets comprising barcoded cDNA in comparison to droplets comprising cDNA which is not barcoded.

0390The skilled in the art will understand that barcoded cDNA may processed differently, because barcoded single cell cDNAs obtained from one cell may be mixed with barcoded single cell cDNAs of another cell, since the cDNAs may be distinguished because of the barcode sequence. Contrary to this, cDNAs that are not barcoded can only be mixed when bound to a particle.

0391Accordingly, in some embodiments, the cDNA is not barcoded, in related embodiments, the cDNAs produced by the reverse transcription of step b) is recovered and then at least one barcode is attached in step c). After attaching a barcode in step c) the barcoded cDNA is further used for, typically, subsequent amplification and sequencing library preparation.

0392In some embodiments, the cDNA is barcoded, in related embodiments, the barcoded cDNAs produced by the reverse transcription of step b) are recovered and further used for, typically, subsequent amplification and sequencing library preparation.

0393Subsequent amplification and sequencing library preparation is as defined herein above in the section <i>"Amplifying and sequencing".</i>

0394Accordingly, in some embodiments, the process of the invention further comprises recovering barcoded single cell cDNAs produced by reverse transcription in at least some of the fused droplets.

0395More precisely, in related embodiments, recovering refers to collecting the microfluidic droplets comprising barcoded cDNA produced by reverse transcription, breaking the microfluidic droplets and separating the barcoded cDNA comprised in the aqueous composition from the oil phase of said microfluidic droplets.

0396Methods to isolate nucleic acids, in particular barcoded cDNA from droplets are known to the skilled in the art and comprise for example, collecting the microfluidic droplets and breaking the microfluidic droplets using typically perfluoro-octanol (v/v emulsion). Then incubating the emulsion obtained in the previous step until the aqueous and oil phase are separated. In one example, the aqueous phase is typically centrifuged for, for example, 10 min at 10,000g at 4°C and the supernatant comprising the cDNA is recovered.

0397In some embodiments, the process further comprises the step of removing unincorporated oligonucleotides.

0398"<u>Removing unincorporated oligonucleotides</u>" comprises contacting the aqueous composition comprising cDNA and eventually unincorporated oligonucleotides with a purification substrate wherein the purification substrate removes unincorporated oligonucleotides. As it will be understood by the skilled in the art, the cDNA submitted to the step of of removing unincorporated oligonucleotides is preferably barcoded cDNA.

0399In some embodiments, the purification substrate comprises beads or particles, which, optionally, form a column. In a further example, unincorporated oligonucleotides are removed by size selection using for example an acrylamide gel.

0400In some embodiments, the step of "<u>removing unincorporated oligonucleotides</u>" comprises contacting the aqueous composition comprising cDNA with an exonuclease to degrade the unincorporated oligonucleotides within the aqueous composition of the at least some of the droplets.

0401In related embodiments, the exonuclease degrades single stranded nucleic acid sequences from the aqueous compositions comprising the cDNA.

0402It will be understood by the skilled in the art that the cDNA, such as the barcoded cDNA, obtained, for example, in step b) or c), is typically present in form of a RNA/DNA complex and thus protected from said exonucleases.

0403In some embodiments, the cDNA comprises one or more nucleotide analogs, as defined herein above, facilitating purification of the cDNA sequences or molecules.

0404As it will be understood by the skilled in the art, in certain embodiments, purified cDNA does not comprise unincorporated oligonucleotides.

0405In some embodiments, the cDNA is further treated with RNAse A.

0406"<u>RNAse A</u>" is an endoribonuclease that specifically degrades single-stranded RNA at C and U residues.

0407In some embodiments, the RNAse A is at a concentration of 10 to 1000µg/µL, preferably 50 to 200 µg/µL, for example at 100µg/µL.

0408In some embodiments, the cDNA is further treated with Proteinase K.

0409"<u>Proteinase K"</u> is a broad-spectrum serine protease and digests proteins, preferentially after hydrophobic amino acids.

0410In some embodiments, the Proteinase K is at a concentration of 0.1 to 5mg/mL, preferably 0.1 to 1 mg/mL, for example at 0.8mg/mL.

0411The recovered cDNA is then further used for, typically, subsequent amplification and sequencing library preparation as defined herein above in the section <i>"Amplifying and sequencing".</i>

0412As explained herein above in the section <i>"oligonucleotides",</i> in embodiments where the at least one oligonucleotide is bound to at least one particle of a first type, reverse transcribing the nucleic acids present in the fused droplet results in single cell cDNA bound to the at least one particle of a first type in form of a RNA/DNA complex.

0413Furthermore, in embodiments where the at least one oligonucleotide does not contain a barcode sequence the cDNA obtained by reverse transcription and bound to said at least one particle of a first type is not barcoded.

0414Accordingly, in a related embodiment, recovering single cell cDNAs herein refers to breaking the microfluidic droplets and separating the single cell cDNA bound to at least one particle of a first type comprised in the aqueous composition from the oil phase of said microfluidic droplets.

0415Methods to break the microfluidic droplets are known to the skilled in the art and include typically the use of perfluoro-octanol (v/v emulsion).

0416The cDNA bound to a particle may be separated from the oil phase using methods known to the skilled in the art. For example, when the particle is a magnetic particle, the cDNA may be separated using a magnet. In another example, when the particle is a hydrogel particle, the cDNA may be separated by sedimentation.

0417Separating the cDNAs bound to at least one particle of a first type from the oil phase results in a pool comprising a plurality of particles of a first type, wherein to each particle cDNA of one cell is bound.

0418In related embodiments, for attaching the at least one barcode sequence, for example in step c), to the single cell cDNA, the process of the invention further comprises a step of distributing single cell cDNAs bound to the at least one particle of a first type into a plurality of reservoirs, wherein at least one particle of a first type, preferably one particle of a first type, to which the cDNA of one single cell is bound, is received in each reservoir. Such a reservoir may be, for example, a well of, typically, a well plate, such as a 96 well plate.

0419In some related embodiments, the process of the invention further comprises the step c) of attaching a barcode sequence to the single cell cDNA obtained in step b), wherein the at least one barcode sequence encodes the identity of said single cell.

0420The at least one barcode sequence is as defined herein above in the section <i>"oligonucleotides".</i> In a preferred embodiment, when the at least one barcode is attached after the transcription step, said at least one barcode sequence is preferably a single stranded nucleic acid. It will be understood by the skilled in the art that one barcode sequence, or more than one barcode sequence may be attached. When more than one barcode sequence is attached, individual barcode sequences attached to each other end up in one final barcode sequence. Those individual barcode sequences may be attached in subsequent cycles.

0421In some embodiments, the step c) of attaching a barcode sequence further comprises attaching a UMI sequence, wherein said UMI sequence is as defined herein above in the section <i>"oligonucleotides".</i>

0422Methods to attach barcode sequences or a UMI sequence are known to the skilled in the art and include, for example, the use of ligases and/or using annealing or a primer extension method.

0423In some embodiments, the step c) of attaching the barcode sequence may be followed by a washing step to remove barcodes that are not attached to the single cell cDNA.

0424In some embodiments, the process further comprises the step of removing unincorporated oligonucleotides. Methods to remove unincorporated oligonucleotides are further described herein above.

0425In embodiments, where the cDNA is attached to at least one particle of a first type, the process further comprises a step of releasing cDNA or barcoded cDNA from the at least one particle of a first type. Methods to release oligonucleotides are described herein above in the section <i>"oligonucleotides"</i> and apply <i>mutatis mutandis</i> to the release of cDNA since the cDNA is bound to the particle via said oligonucleotide.

0426Preferably, the step of releasing the cDNA occurs after attaching the barcode sequence in step c).

0427In some embodiments, the step c) of attaching the barcode sequence is followed by a step of recovering the barcoded cDNA.

0428Recovering the barcoded cDNA herein refers to collecting the barcoded cDNA.

0429The recovered barcoded cDNA is then further used for, typically, subsequent amplification and sequencing library preparation as defined herein above in the section <i>"Amplification and sequencing".</i>

0430Furthermore, in some embodiments, the at least one oligonucleotide was introduced in the RT droplet without a particle or, optionally, released from said particle prior to reverse transcription.

0431Accordingly, it will be understood by the skilled in the art that these embodiments of the process of the invention result, after transcription, in cDNA that is not attached to a particle. Furthermore, in embodiments where the at least one oligonucleotide does not comprise a barcode sequence, said cDNA is not barcoded.

0432Accordingly, in related embodiments, the RT-droplets further comprise a polymer.

0433It will be understood by the skilled in the art, that accordingly, in some embodiments the fused droplets comprise said polymer.

0434In some embodiments, said polymer is in form of a solution.

0435In some embodiments, said polymer is selected from the group consisting of alginic acid, agarose, poly(ethylene glycol) diacrylate, or acrylamide-based gels, such as bis-acrylamide, polyacrylamide, streptavidine acrylamide, poly-N-isopropylacrylamide, or poly N-isopropylpolyacrylamide or mixtures thereof.

0436In a further embodiment, the polymer is functionalized to capture nucleic acids.

0437Methods to functionalize a polymer to capture nucleic acids are known to the skilled in the art and include, but are not limited to, streptavidine, Histidine-tags, biotin, calmodulin, SNAP-tag, Biotin, Acrydite.

0438In a related embodiment, the fused droplets are recovered as described herein above.

0439In related embodiments, the step of recovering fused droplets as defined herein above, further comprises the step of polymerizing the fused droplets.

0440For example, a hydrogel, such as alginic acid may be polymerized by the addition of calcium ions. In other cases, polymerization initiators, also called cross-linker (such as ammonium persulfate and TEMED for acrylamide) may be added to a droplet.

0441It will be understood by the skilled in the art that, when polymerizing the fused droplets, the single cell cDNA produced in step b) is comprised in said polymerized droplets.

0442In some embodiments, polymerization is obtained by cross-linking.

0443In some embodiments the cross-linker is added to the fused droplets, for example, by diffusion.

0444In alternative embodiments, the RT-droplets comprise a cross-linker. It will be understood by the skilled in the art, that accordingly, in the same embodiments, also the fused droplets comprise said cross-linker.

0445In some embodiments, the cross-linker is activated for polymerization.

0446In particular, when the RT droplets comprise a cross-linker said cross-linker requires activation for polymerization.

0447In some embodiment, the cross-linker is activated by heat or UV.

0448In related embodiments, for attaching the barcode in step c) to the cDNA, the process of the invention further comprises a step of distributing polymerized droplets comprising single cell cDNAs in a plurality of reservoirs, wherein, preferably, one polymerized droplet is received in each reservoir. The reservoirs might be the well of, typically, a 96 well plate.

0449It is known to the skilled in the art that small nucleic acids, such as barcode sequences, as well as enzymes, such as ligases, may migrate into polymers as defined herein above.

0450Accordingly, the step c) of attaching the barcode sequence applies to cDNA comprised in said polymer in the same way as described herein above for cDNA attached to a particle. The step of attaching a barcode to the cDNA thus applies as defined herein above in the embodiments referring to cDNA attached to a particle.

0451Accordingly, the barcode may be attached either to the 3' or the 5' end.

0452In some embodiments, the step c) of attaching a barcode sequence further comprises attaching a UMI sequence as defined herein above.

0453In some embodiments, the step c) of attaching the barcode sequence is followed by a washing step to remove barcodes that were not attached to the cDNA.

0454In some embodiments, the process further comprises the step of removing unincorporated oligonucleotides. Removing unincorporated oligonucleotides is as defined herein above.

0455In some embodiments, the process further comprises a step of releasing the barcoded cDNA from the polymerized droplets. cDNA may be released from the polymerized droplets by simple diffusion, photocleaveage or electrophoresis.

0456Preferably, the step of releasing the cDNA occurs after attaching the barcode sequence in step c).

0457In some embodiments, the step c) of attaching the barcode sequence is followed by a step of recovering the barcoded cDNA. The recovered barcoded cDNA is then further used for, typically, subsequent amplification and sequencing library preparation as defined herein above in the section <i>"Amplification and sequencing".</i>

0458"<u>Genotyping</u>" generally refers to the process of determining the nucleic acid sequence of an individual using biochemical methods. Genotyping in context of the present invention refers to determining the nucleic acid sequence of a single cell, in particular of a single cell of interest.

0459A "<u>phenotype</u>" usually refers to the composite of an organism's observable characteristics or traits, such as its morphology, development, biochemical or physiological properties. A phenotype results from the expression of an organism's genetic code, its genotype, as well as the influence of environmental factors and the interactions between the two. In context of the present invention, the phenotype of a single cell refers to the presence or absence of a specific characteristic, such as for example, the presence or absence of a specific antibody, or the presence or absence of a specific antigen or the presence or absence of a specific activity, which might be observed or is identified using, typically, a phenotypic assay.

0460Accordingly, in context of the process for genotyping single cells, single cells present in a plurality of reservoirs, for example, in the reservoirs of, typically, a 96 well plate or in the reservoirs of a microfluidic device, are first phenotyped and then genotyped. The inventors developed a process, wherein, based on the use of specific barcodes in combination with dyes it is possible, to link, after sequencing, the genetic sequence of a barcoded single cell cDNA with the phenotype observed for said single cell.

0461Throughout the instant application, the term "<u>and/or</u>" is a grammatical conjunction that is to be interpreted as encompassing that one or more of the cases it connects may occur. For example, the wording "error detection and/or correction " in the phrase "the sequences allow for error detection and/or correction" indicates that the sequences may allow for error detection and the sequence may allow for error correction or the sequences may allow for error detection or the sequence may allow for error correction.

0462As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references, such as a plurality of the object referred to, unless the content clearly dictates otherwise.

0463Throughout the instant application, the term "<u>comprising</u>" is to be interpreted as encompassing all specifically mentioned features as well optional, additional, unspecified ones. As used herein, the use of the term "<u>comprising</u>" also discloses the embodiment wherein no features other than the specifically mentioned features are present (<i>i.e.</i> "<u>consisting of</u>").

0464In the entire description, features described in one section are entirely applicable to other sections of the instant description. Accordingly, the invention further contains combinations of embodiments described in the different sections of the description.