Single cell analysis

Abstract

The present invention relates to methods for barcoding nucleic acids from single cells and methods for genotyping single cells having a phenotype of interest.

Description

[0001] This application is a divisional application of the invention patent application with the application date of March 15, 2018, application number 202111005542.4, and invention name “Single Cell Analysis”. This application is a divisional application of the invention patent application with the application date of March 15, 2018, application number 201880018953.7, and invention name “Single Cell Analysis”. Technical Field

[0002] The present invention relates to methods for barcoding nucleic acids from single cells and methods for genotyping single cells having a phenotype of interest. Background Art

[0003] Methods for analyzing multiple parameters of single cells in a population 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 population of cells is of broad interest to commercial and academic laboratories. Moreover, such analyses often need to be performed in real time to reveal the dynamic behavior of biological and biochemical processes.

[0004] Thus, technologies that can separate, detect, and quantify individual components of heterogeneous mixtures in a highly parallel manner are needed to meet these challenges. Conventional high-throughput platforms, such as high-density microplates with robotic dispensing systems, have been developed and widely used for high-throughput analysis, such as drug screening. However, they require expensive and cumbersome robotic machines and require sample evaporation and considerable reaction volumes, which wastes precious biological samples or reagents.

[0005] Recently, microfabricated devices have become powerful experimental platforms for carrying out various biological and chemical assays in a high-throughput manner. These technologies generally allow high-throughput analysis of complex samples by distributing large amounts of solutions into many separated compartments or microreactors of picoliter to nanoliter size. However, it is difficult to recover (retrieval) after analyzing individual samples. In addition, mixing reagents in these devices either requires complex structures, or is usually carried out in large quantities before compartmentalization, which can prevent the initial reaction products from being co-localized with their triggering targets.

[0006] One approach is to compartmentalize reactions into discrete micrometer-sized droplets surrounded by an immiscible carrier fluid. Droplet-based microfluidics allows for precise control of fluid mixing, minimizing waste of precious reagents and reducing evaporation and adsorption of molecules on device walls.

[0007] White et al. (Proc Natl Acad Sci USA. 2011Aug 23; 108(34): 13999-4004) described a microfluidic device that can perform RT-qPCR measurements of gene expression of hundreds of single cells per run, performing single cell processing, including cell capture, cell lysis, reverse transcription, and quantitative PCR. However, in an attempt to try smaller reaction volumes, White et al. (Proc Natl Acad Sci USA. 2011Aug 23; 108(34): 13999-4004) performed 300 parallel RT-qPCRs and confirmed that RT was inhibited when the volume was less than 5 nL under the reaction conditions tested. Therefore, the researchers performed RT reactions with 67 nL per cell and further claimed that the combination of RT and qPCR in a single reaction excludes large-scale transcriptome analysis and / or unbiased amplification.

[0008] In addition, DeKosky et al. (Nat Med. 2015 Jan; 21(1): 86-91) have described a method for single-cell mRNA capture using a simple axisymmetric flow focusing device. The RT and PCR reactions of this method are performed in emulsion. This method has been shown to be effective, but a sequential 3-step method is required.

[0009] Along the same line, Eastburn et al. (Anal Chem. 2013 Aug 20; 85(16): 8016-21) performed RT in small droplet volumes, but it took 3-4 steps. He also mentioned the inhibitory effect of cellular proteases on RT in small volumes. To solve this problem, they used protease treatment of cells, dilution of cell lysates, splitting of droplets, and percutaneous injection of RT-PCR reagents.

[0010] Rotem et al. (PLoS One. 2015 May 22; 10(5): e0116328) encapsulated oligonucleotides in a population of droplets and fused them with droplets containing cells, while injecting RT enzyme and buffer percutaneously. This 3-step method produced ~100 pL droplets, allowing 100,000 cells to be encapsulated in 3 h. Although this method relies on single-cell cDNA labeling, transcriptome sequence data comes from aggregates of multiple phenotypically and genotypically unrelated cells.

[0011] Macosko et al. (Cell. 2015 May 21; 161(5): 1202-14) used droplet-based microfluidics to encapsulate cells with lysis reagents and barcoded beads to capture mRNA in 1 nL droplets. However, the beads are used outside the chip to convert the mRNA captured on the beads into cDNA, and it will not be loaded on most of the droplets that have been formed, thereby excluding the analysis of rare cell populations. Step 3 of the method described by Macosko et al. is then devoted to library preparation and amplification.

[0012] International patent application WO2015 / 164212 relates to a method for encapsulating and barcoding single cell nucleic acids. WO2015 / 164212 discloses that the RT of mRNA is strongly inhibited in a volume of less than 3 nL (Example 4 of WO2015 / 164212).

[0013] Labanieh et al. 2015 described a microfluidic method that uses a microfluidic chip to capture, analyze, and recover droplets using buoyancy. However, Labanieh et al. did not explicitly address the analysis of single-cell nucleic acids in which the nucleic acids were reverse transcribed, and therefore the genotype of single cells could not be obtained using this method.

[0014] Patent application US2013 / 0323732 relates to methods and devices for measuring single cells and barcoding single cell cDNA. US2013 / 0323732 does not disclose a method in which droplets are fused in a microfluidic chip.

[0015] In contrast, the present inventors have successfully developed an on-chip microfluidic approach for single-cell nucleic acid barcoding, in which single-cell droplets are captured in individual compartments and further fused with droplets providing a reaction mixture for reverse transcription.

[0016] As described above, the on-chip method developed by the inventors allows the phenotype of single cells to be obtained. Therefore, the inventors have further developed a unique method for coupling phenotypic information (protein expression levels, cell pathway activation / activity, ion channel / GPCR activity) with genotype or epigenetic information, thereby allowing the genotype of single cells with a phenotype of interest to be determined.

[0017] The ability to image the array at various steps in the method allows for more quantitative data per single cell compared to conventional methods and allows for the generation of kinetic data during phenotypic screens, which can then be linked to the genotype of specific single cells.

[0018] This method differs from the prior art known so far, such as Rotem et al., because in Rotem et al., for example, the genomic information is obtained by sequencing an aggregate of multiple phenotypically and genotypically unrelated cells. Therefore, their method does not disclose coupling the phenotypic information of each single cell with the genotypic information.

[0019] International patent application WO2016145409 Al discloses a method in which the phenotype and genotype of a protein of interest can be related. Different from the method of the present inventors, in this method, a barcode is connected to, for example, a protein of interest or its binding partner, and at the same time connected to a nucleic acid encoding the protein of interest. Then, the nucleic acid is reverse transcribed to obtain a barcoded cDNA, and then the barcoded cDNA is sequenced. The protein of interest is phenotyped in parallel by destroying the emulsion and performing a phenotypic assay, such as an ELISA or an assay using an affinity column. For proteins that show a desired phenotype, the barcode sequence can then be analyzed by sequencing. Subsequently, the phenotype is linked to the genotype by matching the barcode of the protein of interest with the cDNA having the same barcode.

[0020] However, one skilled in the art will appreciate that such methods do not allow matching the entire transcriptome of a single cell to its phenotype, or obtaining the genotype of a single cell having a phenotype that is not associated with the presence of a particular protein of interest.

[0021] In contrast, our method allows phenotyping of several single cells in one step, and then genotyping those single cells in parallel by mixing their nucleic acids. Subsequently, the genomic information is linked to the phenotype of the single cell with the phenotype of interest.

[0022] Furthermore, the inventors' methods allow for the determination of any phenotype, including phenotypes that are not necessarily associated with the presence of a single protein of interest, but which may be associated with, for example, altered activity of certain pathways, and which result in any phenotype that can be determined using an assay, e.g., antibody secretion rate, ion channel activity, GPC activation, altered redox potential of a cell. Summary of the invention

[0023] A first aspect of the present invention relates to a method for genotyping a single cell having a phenotype of interest, the method comprising:

[0024] - provide multiple reservoirs, and

[0025] a) for a plurality of reservoirs, providing at least one barcode sequence and at least one dye for each reservoir, wherein the at least one barcode sequence is associated with a color and a concentration of the at least one dye, and acquiring an image of the array to map the color and the intensity of the at least one dye for each reservoir; and

[0026] b) for a plurality of reservoirs, providing at least one single cell for each reservoir and performing a phenotypic assay on the at least one single cell for each reservoir, and acquiring an image, thereby mapping the phenotype of the at least one single cell for each reservoir;

[0027] wherein step a) is performed before step b) or step b) is performed before step a); then

[0028] c) for a plurality of banks, comparing the phenotype of the single cell obtained in step b) with the color of the at least one dye obtained in step a) for each bank;

[0029] associating a color with said intensity; and

[0030] - for a plurality of reservoirs, reverse transcribing the nucleic acid of the at least one single cell for each reservoir to obtain single cell cDNA barcoded with the at least one barcode sequence, the at least one barcode sequence being associated with the color and concentration of the at least one dye; and

[0031] - correlating said genotype with said phenotype of said at least one single cell.

[0032] A second aspect of the present invention relates to a microfluidic method for barcoding single-cell nucleic acids, the method comprising:

[0033] - providing a microfluidic device comprising a chip, the chip comprising at least one microfluidic channel and a plurality of reservoirs,

[0034] - injecting a carrier fluid into an inlet of the microfluidic channel, the carrier fluid comprising a plurality of first type droplets dispersed in the carrier fluid, wherein the first type droplets are 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 a single cell, wherein the single cell comprises a single-cell nucleic acid,

[0035] - for a plurality of reservoirs, a first migration step, wherein at least one droplet of the first type in the plurality of droplets is moved into a reservoir of the plurality of reservoirs by buoyancy,

[0036] - injecting a carrier fluid into an inlet of the microfluidic channel, the carrier fluid comprising a plurality of droplets of a second type dispersed in the carrier fluid, wherein the droplets of the second type are 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,

[0037] - for a plurality of reservoirs, a second migration step, wherein at least a portion of at least one droplet of the second type enters a reservoir of the plurality of reservoirs,

[0038] - for a plurality of reservoirs, said at least one droplet of said first type merges with said at least one droplet of said second type in each reservoir or at its edge, thereby producing a fused droplet,

[0039] - and further comprising the following steps:

[0040] a) for each fused droplet, hybridizing at least some of the single-cell nucleic acid to the at least one oligonucleotide in the fused droplet,

[0041] b) in each fused droplet, reverse transcribing at least some of the single-cell nucleic acid present in the fused droplet, thereby producing single-cell cDNA, and

[0042] c) attaching at least one barcode sequence to the single cell cDNA obtained in step b), wherein the at least one barcode sequence encodes the identity of the single cell,

[0043] or

[0044] - It also includes the following steps:

[0045] a) for each fused droplet, hybridizing at least some of the single-cell nucleic acid from at least one cell to the at least one oligonucleotide in the fused droplet, wherein the at least one oligonucleotide comprises at least one barcode sequence,

[0046] b) in each fused droplet, reverse transcribing at least some of the single-cell nucleic acids present in the fused droplet, thereby generating barcoded single-cell cDNA, wherein the at least one barcode sequence encodes the identity of the single cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1A phenotypic assay based on single cell droplets for identifying antibody-dependent cell-mediated cytotoxicity (ADCC) is shown. Cells (2) producing IgG secrete antibodies that target reporter cells (1) expressing membrane proteins. After the antibody (3) binds to the targeted cells, killer cells (4) are recruited by the Fc receptors of the cells, which will induce cytotoxic activity (10) mediated by binding to the antibody secreted by (2). In some applications, the killer cells are isolated from PMBC or are cell lines known to have cytotoxicity-mediated activity.

[0048] Figure 2 A single cell droplet-based phenotypic assay for identifying complement-dependent cytotoxicity (CDC) antibodies is shown. IgG-producing cells (2) secrete antibodies that target reporter cells (1) expressing membrane proteins. After the antibodies (3) bind to the targeted cells, complement molecules (6) bind to the antibodies inducing cell lysis (10).

[0049] Figure 3 A single cell droplet based phenotypic assay for identifying antibody drug conjugate (ADC) (3) candidates that induce cell lysis, apoptosis, internalization, necrosis is shown. Drug-conjugated antibodies (5) are introduced into different emulsions, one drug per emulsion. Upon binding to the antibody (3), and in case of effective drug activity, the target cells in the given example are lysed (10).

[0050] Figure 4 A single cell droplet-based phenotypic assay for identifying agonist / antagonist antibodies (3) is shown. IgG-producing cells (2) secrete antibodies that target reporter cells (1) expressing membrane proteins. Upon binding of the antibody to the targeted cells (1), several pathways are activated or inhibited, leading to physiological modifications. Depending on the nature of the cells and receptors, these modifications can be used in single-function or multifunctional assays in droplets. Differently, the antagonist antibody (3) will compete with the ligand in the same droplet (7). The readout will then be the activity of the blocking agonist.

[0051] Figure 5A single cell droplet based phenotypic assay for identifying the agonistic activity of a ligand is shown. Image acquisition was performed using an epifluorescence based microscope at 10x magnification. A. Different concentrations of fluorescent dye were added depending on the ligand concentration to distinguish between emulsions containing agonists (higher fluorescent dye concentration) and emulsions without agonists (lower fluorescent dye concentration). B. Cells were labeled with cell markers. After fusion with a second droplet population, containing other substrates for detecting intracellular pathway activation detection and reagents for RT (+ / -PCR), the cells were lysed resulting in diffusion of cell markers inside the droplets. Combining A with B gives information about the assay conditions. C. Finally, a functional readout is given in a third color, which shows the recruitment of downstream agonists that induce activation of intracellular signaling pathways in this case. Droplets above the threshold are considered positive and match the expectations given by condition A+B. This example highlights the feasibility of linking the complex phenotype of secreted antibodies (downstream signaling intracellular cascade activation) with their sequencing information.

[0052] Figure 6 A single cell droplet-based phenotypic assay for identifying antagonistic / blocking antibodies (3) is shown. IgG-producing cells (2) secrete antibodies (3) that target reporter cells (1) expressing membrane proteins. After the antibody binds to the targeted cell (1), the ligand or agonist is gradually released by particles composed of a core and shell (8). The particles are able to release the ligand with kinetics similar to antibody production, thereby enabling competition assays in the droplets. The particles can be composed of a cell growth gel and can generate a ligand gradient, thereby enabling the reporter cells to migrate inside the gel, which is generally referred to as a cell mobility assay (9).

[0053] Figure 7 An embodiment of RT-PCR performed in a microfluidic device for combined phenotypic and genotypic information recovery is shown. In this embodiment, in addition to the RT-PCR reagents, a DNA intercalator or Taq-man probe is added to the mixture. For each cycle, due to the depletion of the intercalator or probe, the copy number increases exponentially until a plateau is reached. Then, an intensity curve is drawn as a function of the cycle, and the value of the cycle threshold (Ct) is calculated. Subsequently, the Ct value is drawn as a function of Log [number of molecules]. The number of molecules for a given sample is calculated according to a standard curve. The standard curve is generated by droplets containing a known amount of molecules, and its Ct is also calculated. Plot these Cts from standard samples as curves of the Log [number of molecules] function, and then calculate the linear regression equation and use it for quantitative analysis of sample droplets containing cells.

[0054] Figure 8An embodiment of a standard procedure for screening IgG-producing cells in droplets is shown. Step 1, involves preparing b cells and reporter cells. The b cell population is initially enriched to increase the relative amount of IgG secretion or production cells in other types of irrelevant populations. Step 2, triggers the co-encapsulation of a single b cell with a single reporter cell or multiple reporter cells. Step 3, incubates the emulsion containing cells so that the IgG-producing cells secrete antibodies in the droplets. Step 4A, sorts the population of interest for the specific phenotypic function (binding ...) induced by the separation of development IgG-specific antibodies. Step 4B, prepares an emulsion containing a reagent for reverse transcription (RT) in droplets in parallel with step 4A. Step 5, the sorted droplets are dispersed in a microfluidic device containing multiple reservoirs. The sorted droplets are captured by buoyancy in the microfluidic device. Step 6, then the captured droplets containing cells and presenting the phenotype of interest are imaged by using a microscope based on epifluorescence or any other device for fluorescence and / or luminescence collection. Step 7, in a subsequent step, the emulsion produced in step 4B is dispersed within the microfluidic device and fused as a single sorting droplet fused with a single RT droplet. Step 8, an incubation step is performed to allow reverse transcription that is ultimately coupled to a PCR reaction. Step 9, after incubation, the microfluidic device is imaged by using an epifluorescence-based microscope or any other device for fluorescence and / or luminescence acquisition. Step 10, the emulsion captured in the microfluidic device is recovered and ultimately subjected to library amplification by PCR. Step 11, the amplified library is sent for sequencing. After the entire process is completed, two steps of data analysis are performed. Related to steps 6 and 9, the images are analyzed according to 3 general steps: correcting the image, detecting each individual well / signal, and signal analysis to give the phenotype of the droplet. Related to step 11, the sequencing data is analyzed according to the following 3 general steps: 1) barcode extraction, clustering and identification of sequence species, 2) antibody sequence characterization and VH / VL pairing, 3) antibody clone assignment and antibody candidate prioritization. In step 12, the combination of these two analyses allows for in-depth characterization of the selected antibodies and links between phenotype and genotype.

[0055] Fig. 9 An image acquired from the microfluidic device containing 1060 buoyancy-based capture units is shown. In this design, two consecutive chambers are used to increase the total number of capture units.

[0056] Fig.10Shown are some images acquired during sorting based on binding of target cells in the presence of cells of interest. A. Bright field acquisition of two capture units containing two sorting droplets loaded with one target cell and one cell of interest. B. Image acquired using a TRITC filter. The use of fluorescent molecules within the droplets allows for detection in microfluidic devices. C. Image acquired using a Cy5 filter, showing localization of the detection reagent on the target cell.

[0057] Fig.11 Examples and controls for library preparation are shown. After two-step PCR reactions (PCR1 and PCR2), cDNA was amplified into an Illumina-based sequencing library. PCR2 typically showed amplification products of ~550 bp for VH and VL.

[0058] Fig.12 Several steps are shown that take place during NGS sequencing analysis. One of them consists of pairing the VH sequence of a single cell IgG producing cell with the VL. The graph shows the number of pairs (abundance of reads) used for VH and VL pairing. DETAILED DESCRIPTION

[0059] definition

[0060] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by one of ordinary skill in the art.

[0061] Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0062] In general, nomenclatures and techniques associated with 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.

[0063] "Microfluidics" generally refers to channels in which fluids circulate with dimensions less than 1 millimeter and including, for example, from 1 μm to 1 mm.

[0064] Microfluidics generally refers to the use of microfluidic channels with dimensions less than 1 mm to manipulate small amounts of fluid (10 -4 Up to 10 -18 Those microfluidic channels are generally contained in a microfluidic device, more particularly in a microfluidic chip of a microfluidic device. The microfluidic device used in the background of this application is further described in the "Microfluidic Device" section below in this application.

[0065] The "process" of the present invention may also be referred to as a method.

[0066] Standard 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 the manufacturer's instructions or according to the usual methods in the art or as described herein. Unless specifically indicated to the contrary, the implementation of the present disclosure will employ conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA technology within the technical scope of the art, many of which are described below for illustrative purposes. Such techniques are fully described in the literature. See, for example, Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: APractical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic acid AcidHybridization (B.Hames&S.Higgins, eds., 1985); Transcription and Translation (B.Hames&S.Higgins, eds., 1984); Animal Cell Culture (R.Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984).

[0067] The terminology and 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.

[0068] As used in this application, the term "nucleic acid" generally refers to at least one molecule or strand of DNA, RNA, miRNA, or a derivative or mimetic thereof, which comprises at least one nucleobase, such as 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 "nucleic acid" includes the term "oligonucleotide".

[0069] "RNA" in this application refers to but is not limited to functional RNA, such as mRNA, tRNA, rRNA, catalytic RNA, siRNA, miRNA and antisense RNA. In a preferred embodiment, RNA refers to mRNA.

[0070] As will be appreciated by those skilled in the art, the description of a single strand also defines the sequence of the complementary strand. Thus, nucleic acid also includes the complementary strand of the described single strand. Therefore, the term nucleic acid includes complementary DNA. It will also be appreciated by those skilled in the art that a lot of nucleic acid variants can be used for the same purpose as a given nucleic acid. Thus, nucleic acid also includes substantially identical nucleic acids and their complements. It will also be appreciated by those skilled in the art that a single stranded nucleic acid (such as a primer) can hybridize with a target sequence under hybridization conditions (preferably under stringent hybridization conditions). Thus, nucleic acid also includes a primer that hybridizes with a target sequence under hybridization conditions.

[0071] The term "oligonucleotide" refers to a molecule of at least one length of about 3 to about 500 core bases. For example, the length of the oligonucleotide can be at least 3 core bases, at least 10 core bases, at least 30 core bases, at least 50 core bases, at least 100 core bases, at least 300 core bases or at least 400 core bases. In some cases, the length of the oligonucleotide can be no more than 500 core bases, no more than 300 core bases, no more than 100 core bases, no more than 50 core bases, etc. Any combination of these is also possible, for example the length of the oligonucleotide can be between 3 to 300 core bases, preferably between 3 to 200 core bases, more preferably between 3 to 100 core bases.

[0072] These definitions refer to at least one single-stranded molecule, but in some embodiments also include at least one additional strand that is partially, substantially or completely complementary to the at least one single-stranded molecule. Thus, in some embodiments, the definitions refer to double-stranded molecules.

[0073] Thus, in some embodiments, a nucleic acid refers to at least one double-stranded molecule that comprises one or more complementary strands, or a "complement" of a specific sequence of strands of the molecule.

[0074] As used in the present application, "gene" can be a genomic gene comprising transcription and / or translation regulatory sequences and / or coding regions and / or non-translated sequences (e.g., introns, 5'- and 3'-non-translated sequences). The coding region of a gene can be a nucleotide sequence encoding an amino acid sequence or a functional RNA (e.g., tRNA, rRNA, catalytic RNA, siRNA, miRNA and antisense RNA). A gene can also be an mRNA or cDNA corresponding to a coding region (e.g., exons and miRNA), which optionally comprises a 5'- or 3'-non-translated sequence connected thereto. A gene can also be an amplified nucleic acid molecule produced in vitro, which comprises all or part of a coding region and / or a 5'- or 3'-non-translated sequence connected thereto.

[0075] As used herein, the term "stringent conditions" or "highly stringent conditions" corresponds to conditions suitable for producing binding pairs between nucleic acids with a determined level of complementarity, but not suitable for forming binding pairs between nucleic acids showing complementarity below the determined level. Stringent conditions are a combination of hybridization and washing conditions and are sequence-dependent. These conditions can be improved according to methods well known to those skilled in the art (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). Under normal circumstances, highly stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm), preferably at a temperature close to the Tm of a complete base-paired double helix (Andersen, Nucleic acid Hybridization, Springer, 1999, p.54).

[0076] Hybridization procedures are well known in the art and are described, for example, in Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Seidman, JG, Smith, JA, Struhl, K. eds. (1998) Current protocols in molecular biology. VB Chanda, series ed. New York: John Wiley & Sons.

[0077] High stringency conditions typically involve hybridization in 5x SSC / 5x Denhardt solution / 1.0% SDS at about 50°C to about 68°C, wherein the temperature typically corresponds to the highest melting temperature TM of the nucleic acid hybridizing to the target sequence, and washing in 0.2x SSC / 0.1% SDS at about 60°C to about 68°C. For example, in the context of the present invention, the primer sequence contained in the at least one oligonucleotide is typically hybridized to a complementary nucleic acid (e.g., a complementary RNA sequence) at about 50°C to about 68°C, typically in a reservoir or droplet or droplets (such as a so-called fusion droplet or droplets). Thus, in one example, the fusion droplet or the reservoir further comprises a reverse transcriptase composition, and optionally a lysis composition, wherein the reverse transcriptase composition is defined in the "Reverse Transcription" section below and the lysis composition is defined in the "Cell Lysis" section below.

[0078] The term "antibody" refers to immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, i.e., molecules containing antigen binding sites that immunospecifically bind to antigens. Thus, the term antibody includes not only complete antibody molecules, but also antibody fragments and antibody variants, including derivatives such as humanized antibodies. In conventional antibodies, two heavy chains are connected to each other by disulfide bonds, and each heavy chain is connected to a light chain by a disulfide bond. There are two types of light chains lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes), which determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA and IgE. Each chain contains different sequence domains. The light chain includes two domains: a variable domain (VL) and a constant domain (CL). The heavy chain includes 4 domains: 1 variable domain (VH) and 3 constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant region domains of the light chain (CL) and the heavy chain (CH) confer important biological properties, such as antibody chain binding, secretion, trans-placental movement, complement binding, and binding to Fc receptors (FcRs). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and is composed of the variable portions of one light chain and one heavy chain. The specificity of an antibody lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is composed of residues primarily from the hypervariable region or the complementary determining region (CDR). Occasionally, residues from non-hypervariable regions or framework regions (FR) affect the structure of the entire domain and thus the binding site. The complementary determining region (CDR) refers to the amino acid sequence that together defines the binding affinity and specificity of the natural Fv region of the natural immunoglobulin binding site. The light chain and heavy chain of an immunoglobulin each have three CDRs, referred to as L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, the antigen binding site includes six CDRs, which comprise a set of CDRs from each heavy and light chain V region. Framework regions (FRs) refer to amino acid sequences between CDRs, i.e., those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved between different immunoglobulins of a single species, as defined by Kabat et al. (Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1991).

[0079] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10): 1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Thus, the term antibody further refers to single-chain antibodies, such as camelid antibodies or nanobodies or VHHs.

[0080] The term "T cell receptor" herein refers to an antigen recognition molecule present on the surface of a T cell (T lymphocyte). This definition explicitly includes the understanding of the term as known in the art, and includes, for example, a receptor comprising or consisting of a disulfide-linked heterodimer of a hypervariable α or β chain expressed on the cell membrane as a complex with an invariant CD3 chain, or a receptor comprising or consisting of a variable γ and δ chain expressed on the cell membrane as a complex with CD3 on a subset of T cells.

[0081] "Antibody genes" and "T cell receptor genes" undergo a unique genetic recombination mechanism called V(D)J recombination, which occurs only in developing lymphocytes at the early stages of T cell and B cell maturation. It involves somatic cell recombination and results in the production of highly diverse repertoires of antibodies / immunoglobulins (Ig) and T cell receptors (TCR) on B cells and T cells, respectively.

[0082] In this application, the term "plurality" refers to any number greater than 1 related object, for example, greater than 500, greater than 1000, greater than 2000, greater than 5000, greater than 100000, for example, 1 to 100000, for example, 1 to 10000, 1 to 10000, 10 to 1000, 50 to 1000, 100 to 1000, 10 to 500 related objects.

[0083] Microfluidic devices

[0084] The microfluidic method of the present invention is performed on a microfluidic device comprising a chip.

[0085] "Microfluidic chip" generally refers to a group of microchannels made by grinding, etching, ablation or molding a material (glass, silicon or polymer, such as PDMS, PMMA or COC). A microfluidic chip generally includes a substrate and a support, which together define at least one channel.

[0086] In some embodiments of the invention, a microfluidic device used in the context of the present invention comprises a chip comprising a plurality of reservoirs.

[0087] In some embodiments of the invention, a microfluidic device used in the context of the present invention comprises a chip comprising at least one microfluidic channel and a plurality of reservoirs.

[0088] In a related embodiment, at least one microfluidic channel extends along a longitudinal direction (X) between an inlet and an outlet.

[0089] In a related embodiment, each of the plurality of reservoirs extends along a height direction (Z) that forms a non-zero angle with the longitudinal direction (X), and each reservoir extends in the longitudinal direction (X) to at least one channel.

[0090] Thus, in some embodiments, a microfluidic device comprises a chip comprising:

[0091] - at least one microfluidic channel extending along a longitudinal direction (X) between an inlet and an outlet,

[0092] - A plurality of reservoirs, wherein each reservoir extends along a height direction (Z) forming a non-zero angle with the longitudinal direction (X) and each reservoir opens into at least one channel.

[0093] In some embodiments, "at least one microfluidic channel" 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.

[0094] In some embodiments, the non-zero angle formed between the height direction (Z) and the longitudinal direction (X) is from 85° to 95°, preferably 90°.

[0095] The 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 at least one fluid to flow along a main direction from the inlet to the outlet.

[0096] The inlet of the channel may be connected to at least one reservoir, for example 1 to 5, preferably 1 to 4, more preferably 1 to 3, such as 1, 2 or 3 reservoirs for the liquid flowing in the channel.

[0097] The outlet of the channel may be connected to at least one reservoir (preferably 1 or 2 reservoirs) to collect fluid from the channel.

[0098] In some embodiments, the chip is a droplet array, more particularly a floating droplet array.

[0099] In some embodiments, the microfluidic device further comprises at least one injection device for controlling the flow rate of at least one fluid.

[0100] In this article, "at least one fluid" can refer to at least 1, at least 2, at least 3, at least 4, at least 5 or more than 5 fluids. The "at least one fluid" injected at the inlet and the "at least one fluid" collected at the outlet can be the same fluid or different fluids.

[0101] The injection device may comprise a pressure controller or a syringe driver.

[0102] In some embodiments, the at least one fluid at the inlet refers to 1, 2, 3 or 4 fluids, such as 3 fluids.

[0103] Thus in one example, one fluid contains, for example, a first type of droplets, a second fluid contains, for example, a second type of droplets, and a third fluid removes a plurality of first or second type of droplets and clears them from the chamber. The first type and the second type of droplets are as defined herein below in the context of the inventive method. According to the example, the microfluidic device further comprises an injection device for controlling the flow rates of the three fluids.

[0104] In some embodiments, the device comprises at least one electrical system, preferably one electrical system, adapted to induce an electric field in a specific region of the chip. The at least one electrical system may include a generator, an anode and a cathode.

[0105] In some embodiments, microfluidic devices in the context of the present invention are used in device assemblies.

[0106] The device components may further comprise a cell sorter or a unit for generating droplets, such as droplets of the first type or the second type, such as single-cell droplets or RT droplets, as further defined below.

[0107] Thus, in an alternative embodiment, the chip may comprise units for producing a first type or a second type of droplets (eg single-cell droplets or RT droplets, as further defined below).

[0108] These droplets are then injected into at least one microfluidic channel through an inlet.

[0109] "Multiple banks" herein refers to any number of banks. The number of banks and therefore a plurality of banks preferably refers to from 1 to 100000 banks, such as 1 to 10000 banks, such as 1 to 8000, 10 to 7000, 10 to 6000, 10 to 5000, 10 to 4000, 10 to 3000, 10 to 2000, 10 to 1000, such as 1000 to 10000, 1000 to 9000, 2000 to 8000, 3000 to 7000, 4000 to 6000, such as 4500 to 5500.

[0110] In the context of the present invention, reference may be made to one reservoir of a microfluidic device. However, a person skilled in the art will appreciate that the method described for one reservoir may be applied in parallel to a plurality of reservoirs present in the chip used.

[0111] In some embodiments, the reservoirs of the chip are cylindrical. In some embodiments, the reservoir sizes of the plurality of reservoirs are uniform.

[0112] It will be understood by those skilled in the art that a microfluidic device (more particularly a chip of a microfluidic device) can be characterized by its geometric parameters, such as the diameter (d 储库 ), the depth of the reservoir (h 储库 ), the height of the injection channel (h 通道 ), the width of the injection channel (w 通道 ) and the space (x) between individual reservoirs in the plurality of reservoirs.

[0113] Droplets

[0114] A "droplet" is usually a volume indicator. In the context of the present invention, a "droplet" refers to a separate portion of a first fluid surrounded by a second fluid. It is worth noting that a droplet is not necessarily spherical, but may assume other shapes, depending on, for example, the external environment.

[0115] The term "droplet" used in the context of the methods of the present invention includes a first type of droplet, a second type of droplet (eg, a single-cell droplet or a RT droplet) and a fused droplet, or a plurality of such droplets.

[0116] Methods for preparing and injecting droplets are well known in the art and will be further explained in the following section "Preparation of Droplets" of this application. For the preparation of droplets, it will be understood by those 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 for preparing microfluidic droplets can be selected to obtain a plurality of microfluidic droplets having a specific volume.

[0117] In some embodiments, in the context of the methods of the present invention, the droplets (particularly the first type of droplets or the second type of droplets) are preferably substantially monodisperse.

[0118] "Monodisperse" herein refers to droplets having substantially the same shape and / or size.

[0119] As described herein, and as is known in the art, the droplets follow a shape and size distribution. Thus, the droplets can have a uniform distribution of cross-sectional diameters, i.e., the droplets can have a diameter or volume distribution 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 that is 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 droplets having a uniform distribution of cross-sectional diameters are disclosed in International Patent Application No. PCT / US2004 / 010903, entitled "Formation and Control of Fluidic Species," filed by Link et al. on April 9, 2004 (published as WO 2004 / 091763 on October 28, 2004), which is incorporated herein by reference. It will be understood by those skilled in the art that when reference is made to the volume or size of a droplet in this application, the volume or size refers to the average volume or size of a plurality of droplets. One of ordinary skill in the art will be able to determine the average diameter of a population of droplets, for example using laser scattering or other known techniques. Droplets formed in the context of the present invention may be spherical, or in some cases non-spherical. The diameter of a droplet, particularly a non-spherical droplet, may be taken as the diameter of a perfect mathematical sphere having the same volume as a non-spherical droplet.

[0120] A "droplet" or "plurality of droplets", such as "plurality of RT droplets" or "plurality of single-cell droplets", has an average volume of less than 5 nL, such as less than 4 nL, less than 3 nL, preferably less than 3 nL. In some embodiments, the plurality of microfluidic droplets have an average volume of less than 3 nL, less than 2.5 nL, less than 2 nL, less than 1.5 nL, less than 1 nL, less than 0.5 nL, such as 0.1 nL to 3 nL, 0.5 nL to 3 nL, 1 nL to 3 nL, typically 10 pL, 20 pL, 30 pL, 50 pL, 0.1 nL, 0.5 nL, 1 nL, 1.2 nL, 1.4 nL, 1.6 nL, 1.8 nL, 2.0 nL, 2.2 nL, 2.4 nL, 2.6 nL, 2.8 nL, 3 nL.

[0121] In some embodiments, droplets of the plurality of microfluidic droplets have an average volume of 1 pL to 5000 pL, 10 pL to 5000 pL, or 10 pL to 3000 pL.

[0122] In some embodiments, droplets of the plurality of microfluidic droplets have an average volume equal to or less than 1 nL.

[0123] Thus, a "fused droplet" or "plurality of fused droplets" has an average volume of less than 10 nL. In some embodiments, the plurality of fused droplets have an average volume of less than 9 nL, less than 8 nL, less than 7 nL, less than 6 nL, less than 5 nL, less than 4 nL, less than 3 nL, less than 2 nL, less than 1 nL, less than 0.5 nL, for example, 0.1 nL to 10 nL, 0.1 nL to 8 nL, 0.1 nL to 6 nL, 0.1 nL to 5 nL, such as 0.1 nL to 3 nL, 0.5 nL to 5 nL, 0.5 nL to 3 nL, 1 nL to 3 nL, typically 0.1 nL, 0.5 nL, 1 nL, 1.2 nL, 1.4 nL, 1.6 nL, 1.8 nL, 2.0 nL, 2.2 nL, 2.4 nL, 2.6 nL, 2.8 nL, 3 nL, 4 nL or 5 nL, for example, 11 pL to 8000 pL.

[0124] In some embodiments, the plurality of droplets of the first type or the second type (preferably the first type) have a height less than the injection channel height (h 通道 )’s average diameter.

[0125] In some embodiments, the plurality of droplets of the first type or the second type (preferably the first type) have an injection channel height (h 通道 ) 30% to 100%, for example, injection channel height (h 通道 ) 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 92% to 98%, 94% to 98%, for example 95% of the average diameter.

[0126] As will be understood by those skilled in the art, in particular, when at least one first type of droplet in the plurality of droplets is moved into one of the plurality of reservoirs by buoyancy, the plurality of first type of droplets have an injection channel height (h ) as described above. 通道 )30% to 100% of the average diameter.

[0127] In some embodiments, the plurality of droplets of the first type or the second type (preferably the first type) have a reservoir diameter (d 储库 ) 60% to 95%, such as 70% to 90%, 75% to 85% or 75% to 80%, preferably 75% to 80% of the average diameter.

[0128] In some embodiments, the plurality of second type droplets have a height greater than the injection channel height (h 通道 ), for example, the average diameter is 100% to 250%, 120% to 250%, 150% to 250%, 200% to 250%, preferably 200% to 250%.

[0129] As will be understood by those skilled in the art, when at least a portion of a droplet of the second type enters one of the plurality of reservoirs during the migration step due to the difference in surface energy, the plurality of droplets of the second type have a height greater than the injection channel height (h) as described herein. 通道 )’s average diameter.

[0130] In some embodiments, the plurality of droplets of the second type have an average diameter that is smaller than or as large as the droplets of the first type.

[0131] In some preferred embodiments, a plurality of droplets of the first or second type (preferably the first type) have an average volume of 1 pL to 5000 pL, typically 1 pL to 4000 pL, 1 pL to 3000 pL, for example 1 pL to 500 pL, 1 pL to 400 pL, 1 pL to 300 pL, 1 pL to 200 pL or 1 pL to 100 pL, preferably 1 pL to 100 pL.

[0132] In certain embodiments, the plurality of droplets of the second type has an average volume that is equal to or greater than an average volume of the plurality of droplets of the first type.

[0133] In some preferred embodiments, the plurality of second type droplets have an average volume of 10 pL to 5000 pL, 10 pL to 4000 pL, 10 pL to 3000 pL, for example 100 pL to 4000 pL, 100 pL to 3000 pL, 100 pL to 2000 pL or 100 to 1000 pL, preferably 100 pL to 1000 pL.

[0134] In some embodiments, the plurality of droplets of the second type have a reservoir depth (d 储库 ), for example, 150% to 250%, 170% to 230%, 180% to 220%, 190% to 210%, for example, with a reservoir depth (d 储库 ) is an average diameter of 200%.

[0135] In some embodiments, the plurality of droplets of the second type have a reservoir diameter (d 储库 ), such as 80% to 120%, 90% to 120%, 100% to 120%, 95% to 115%, 95% to 110% of the reservoir diameter, preferably the reservoir diameter (d 储库 ) of 100% to 120% of the average diameter.

[0136] In some embodiments, when the second type of droplet portion enters the reservoir, the plurality of second type of droplets have a diameter greater than the reservoir diameter (d 储库 )’s average diameter.

[0137] In the context of the present invention, a microfluidic droplet or a plurality of microfluidic droplets comprises an aqueous composition.

[0138] In the context of the present invention, an "aqueous composition" in the case of single-cell droplets is typically appropriate for the cells used and typically comprises a buffer solution as defined herein below. In the case of RT droplets, the aqueous composition may also comprise, for example, a reverse transcriptase, a reverse transcription composition and / or a lysis composition.

[0139] Preparation of droplets

[0140] In the context of the method 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 at least one microfluidic channel, preferably one microfluidic channel.

[0141] The first type of droplets are single-cell droplets or RT droplets, and the characteristics of these droplets are further defined in the sections "Microfluidic Methods for Barcoding Single-Cell Droplets", "Single Cell" and "Reverse Transcription" below in this article, respectively, wherein when the first type of droplets are single-cell droplets, the second type of droplets are RT droplets, or when the first type of droplets are RT droplets, the second type of droplets are single-cell droplets.

[0142] Those of ordinary skill in the art will be aware of techniques for preparing microfluidic droplets. Techniques for encapsulating cells in microfluidic droplets are described, for example, in U.S. Patent Nos. 7,708,949, 8,337,778, 8,765,485 or International Patent Application Publication Nos. WO 2004 / 091763 and WO 2006 / 096571, PCT / EP2016 / 080341, each of which is incorporated herein by reference. Techniques for preparing RT droplets comprising reverse transcriptase and at least one oligonucleotide can be easily derived by those skilled in the art from the same disclosure (e.g., PCT / EP2016 / 080341).

[0143] In one example, single cell droplets or RT droplets are prepared before being injected into a separate microfluidic device. In this example, single cell droplets or RT droplets are typically prepared in a microfluidic device having an inlet for a droplet carrier oil (carrier fluid) and an additional inlet for a droplet aqueous phase component. For the carrier oil, typically, a fluorinated oil (e.g., HFE-7500) containing, for example, 0.75% (w / w) surfactant (PFPE-PEG-PFPE triblock copolymer containing two perfluoropolyether blocks (PFPE) and one polyethylene glycol (PEG) block) is typically used. Surfactants are typically used to prevent droplet coalescence, and their dosage can be adjusted, for example, 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 hydrocarbon-based fluids (e.g., mineral oil, hexane, etc.), silicone oils, and other types of oils can be successfully used.

[0144] For preparation of single cell fluidics, one inlet is used for the droplet carrier oil and typically an additional inlet is used to deliver a suspension of dissociated cells.

[0145] For preparing RT droplets, in one example, one inlet is used for the droplet carrier oil, and the other two inlets are typically used to deliver (1) reverse transcriptase and (2) at least one oligonucleotide.

[0146] In some embodiments, for preparing RT droplets, for example, in the context of the present method, RT droplets can be fused with other droplets further containing components, for example, at least one barcode sequence and at least one dye. Methods for merging flowing droplets are well known to those skilled in the art and are described, for example, in WO2010128157 Al.

[0147] As will be appreciated by one skilled in the art, for example, in the case of single-cell droplets, the number of cells encapsulated in one droplet and the size of the droplet follow a probability distribution, such as a Poisson or Gaussian distribution, and depend on the cell concentration, the geometry of the main channel, the injection parameters of the cell suspension and the carrier fluid.

[0148] Thus, the parameters can be adjusted to obtain droplets with 1 or 0 cells therein, thereby avoiding droplets containing a few cells.

[0149] Thus, in the context of the present invention, at least some of the single-cell droplets contain a single cell.

[0150] In the context of the present invention cells comprise nucleic acids, and thus a person skilled in the art will appreciate that in the context of the present invention a single-cell droplet contains a single cell and thus contains single-cell nucleic acid.

[0151] Similar to the preparation of single cell droplets, it will be understood by those skilled in the art that in the case of RT droplets, the encapsulation of the reverse transcriptase and the at least one oligonucleotide follows a probability distribution, such as 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 reverse transcriptase, the at least one oligonucleotide and the injection parameters of the carrier fluid. In addition, in the same embodiment, the at least one oligonucleotide contained in the RT droplet is bound to at least one particle of the first type.

[0152] Therefore, the number of particles of the first type encapsulated in one droplet also follows a probability distribution, such as a Poisson distribution, and depends on the particle concentration, the geometry of the main channel, the particle suspension and the injection parameters of the carrier fluid.

[0153] Thus, the parameters may be adjusted to obtain droplets having 0, 1 or 2 particles therein. Thus, in a certain embodiment, at least some of the RT droplets preferably contain one particle of the first type.

[0154] The "carrier fluid" is immiscible with the aqueous solution of the droplets.

[0155] The "carrier fluid" used to prepare the droplets is well known to those skilled in the art and is typically a fluorinated oil.

[0156] Thus, in some embodiments, the carrier fluid is a fluorinated oil, such as HFE-7500.

[0157] Furthermore, in order to prevent coalescence of the droplets, the carrier fluid may also contain a surfactant.

[0158] Thus, in some embodiments, the carrier fluid further comprises a surfactant.

[0159] The surfactant is typically 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) of a surfactant, such as a PFPE-PEG-PFPE triblock copolymer containing two perfluoropolyether blocks (PFPE) and one polyethylene glycol (PEG) block.

[0160] The surfactant is preferably highly soluble in the fluorinated fluid and nearly insoluble in the aqueous phase.

[0161] However, since the RT droplets and single-cell droplets fuse in a later step, the choice and concentration of the surfactant used to prepare the droplets preferably does not prevent fusion of the droplets.

[0162] Single Cell

[0163] As described in the section "Preparation of droplets" above, the encapsulation conditions (conditions for preparing single-cell droplets) are preferably selected in such a way that the single-cell droplets contain 0 or 1 cell. In order to increase the percentage of droplets containing single cells, the multiple single-cell droplets can be screened and sorted before the step of injecting the multiple single-cell droplets.

[0164] Furthermore, in some embodiments, the plurality of single-cell droplets can be screened for a phenotype of interest prior to injection into the plurality of single-cell droplets.

[0165] According to an alternative embodiment, after injecting the plurality of single-cell droplets, the plurality of single-cell droplets may be screened for a first phenotype and / or a second phenotype of interest.

[0166] An example of a phenotype of interest is the detection of a biological response. Among others, the detection of an immune response can be monitored by detecting ADCC (antibody-dependent cell-mediated cytotoxicity) (3) induced by the secretion of antigen-specific antibodies (see Figure 1 ).

[0167] In biological events, immune responses can be monitored by measuring complement-dependent cytotoxicity (CDC) (6) induced by the secretion of antigen-specific antibodies (see Figure 2 ).

[0168] In biological events, the drug effect can be monitored by detecting ADC (antibody drug conjugate) induced by the secretion of antigen-specific antibodies (see Figure 3 ).

[0169] In biological events, including testing drug function, such as secretion of antigen-specific antibodies and induction of agonist / antagonist antibodies (see Figure 4 , 5 ).

[0170] In biological events, including testing drug function, such as competition assays (see Figure 6 ).

[0171] In biological events, including detection of drug function, such as detection of cell migration of reporter cells mediated by antibody secretion (see Figure 6). Such an assay can be used to track behavioral changes in reporter cells within the droplets. Target cells (1) expressing an antigen of interest and loaded with unreleased detectable markers will be co-encapsulated with IgG producing cells (2) and particles as single cell droplets. The droplets will be incubated under conditions that allow antibody secretion. The secreted specific antibodies will be relocated to the target cells. At the same time, the ligand / agonist diffuses from the particles with dynamics similar to IgG production and produces a ligand gradient. The reporter cells will migrate into the particles under the action of the ligand concentration (9). In the case of blocking antibodies (3), the progress of the reporter cells is changed. Then, migration can be used to evaluate the blocking / neutralizing activity of the antibodies.

[0172] Therefore, in some embodiments, the single-cell droplets are screened and sorted prior to injection into the plurality of single-cell droplets.

[0173] Microfluidic cell sorting technology is well known to those skilled in the art and is described, for example, in Wyatt Schields, C. et al. (Lab Chip. 2015 February 16; 15(5): 1230-1249).

[0174] Examples of microfluidic cell sorting techniques include, but are not limited to, electrophoresis, dielectrophoresis (DEP), electroosmotic flow, acoustophoresis, optical manipulation, mechanical systems, magnetophoresis, electrokinetic mechanisms, optical tweezers, and passive cell sorting. Some methods may also combine several of these techniques.

[0175] Passive cell sorting in this context refers to methods that rely on intrinsic differences and use inertial forces, hydrodynamic diffusion, deterministic lateral displacement, filtration, transient cell adhesion, and cell immobilization to sort cells.

[0176] In a preferred embodiment, prior to injecting the plurality of single-cell droplets, the single-cell droplets are sorted using acoustophoresis or by dielectrophoresis.

[0177] In one example, acoustophoresis (particularly surface acoustic waves) is preferably used to move selected droplets into different sorting bins, such as described in US20130213488 Al.

[0178] In a further preferred embodiment, single cell droplets are sorted prior to injection, using methods such as those described in WO2016059182.

[0179] As is known to those skilled in the art, some of these methods (such as magnetophoresis, acoustophoresis or electrokinetic mechanisms) may require labeling of single cells. However, the type of labeling and whether labeling is required may depend on the cells to be analyzed. For example, for magnetophoresis, for example, cells can be labeled using magnetic particles, however, for example, red blood cells can be sorted based on their natural iron content. Alternatively, in some instances, ferrofluids or paramagnetic solutions can be used to manipulate some types of cells.

[0180] In most embodiments, the single-cell droplet also comprises a fluorescent dye.

[0181] As used herein, "fluorescent dye" refers to a fluorescent substance that can re-emit light when excited by light.

[0182] In some embodiments, for example, when used in the context of cell sorting, the fluorescent dye is conjugated to an antibody that recognizes a target feature on or within a cell; the dye may also be attached to a chemical entity that has an affinity for the cell membrane or other cellular structure. In some embodiments, the fluorescent dye recognizes a cellular structure.

[0183] In some embodiments, fluorescent dyes include, but are not limited to, reactive and conjugated dyes, nucleic acid dyes, cell function dyes, and fluorescent proteins or fluorescent nanoparticles.

[0184] "Reactive and conjugated dyes" include, but are not limited to, xanthenes (eg, fluorescein), rhodamine, coumarin, and cyanine dyes or their derivatives such as Alexa fluor dyes.

[0185] "Nuclear stains" include, but are not limited to, Sytox dye, DRAQ7, propidium iodide (PI), and 7-aminoactinomycin D (7-AAD).

[0186] "Amine dyes" bind to amino groups on cellular proteins. Those dyes bind to proteins on the cell surface or to proteins inside dead cells.

[0187] "Cellular function dyes" are dyes that become fluorescent under specific metabolic conditions of the cell and include, for example, calcein. 2+ When bound, calcein becomes fluorescent.

[0188] "Fluorescent proteins" are well known to those skilled in the art and include, but are not limited to, GFP or EBFP.

[0189] "Fluorescent nanoparticles" include, but are not limited to, PAN particles, for example.

[0190] As will be appreciated by those skilled in the art, some of the sorting techniques mentioned above may require the introduction of particles into single-cell droplets.

[0191] Thus, in some embodiments, the single-cell droplet may further comprise at least one second type of particle.

[0192] In some embodiments, at least one second type of particle herein refers to at least 1, at least 2, at least 3, such as more than 1, such as 1 to 10, 1 to 5, 1 to 3 particles.

[0193] In some embodiments, the second type of particles are fluorescent.

[0194] In some embodiments, the second type of particles is selected from the group consisting of elastomeric particles, hydrogel particles, polymer particles or magnetic particles, preferably hydrogel particles or magnetic particles, wherein magnetic particles and hydrogel particles are defined in the following section "Oligonucleotides" of this application.

[0195] In some embodiments, the magnetic particles are paramagnetic particles or superparamagnetic particles.

[0196] The "elastomer particles" in the present application refer to, for example, silicone elastomer particles.

[0197] In some embodiments, the second type of particle is functionalized so that it can attach other molecules, such as proteins, nucleic acids, or small molecules. Methods for functionalizing particles are well known to those skilled in the art.

[0198] In some embodiments, the second type of particle is functionalized using antibodies.Antibodies can be used to functionalize the second type of particle by using, for example, streptavidin-biotin linkage, as described in the "RT droplets" section below in the context of the first type of particle.

[0199] In one embodiment, the antibody is directed against a component of a single cell.

[0200] In the context of the present invention, "cell" has its ordinary meaning as used in biology, e.g., a cell refers to an autonomous self-replicating unit that can exist as a functionally independent unit of life (e.g., a unicellular organism) or as a subunit of a multicellular organism (e.g., in plants and mammals), which is specialized in carrying out a specific function that enables the organism as a whole. However, "cell" may also refer to a dormant cell, which is generally still capable of cell division when a mitotic stimulus is applied.

[0201] In some embodiments, the cell refers to a prokaryotic cell or a eukaryotic cell, preferably a eukaryotic cell.

[0202] The defining characteristic that distinguishes "eukaryotic cells" from "prokaryotic cells" is that eukaryotic cells have membrane-bound organelles, especially the nucleus, which contains the genetic material and is surrounded by a nuclear membrane.

[0203] In the context of the present invention, "eukaryotic cells" are selected from the group consisting of mammalian cells, plant cells and fungal cells, preferably mammalian cells.

[0204] "Mammal" in this application refers to any mammal, including humans, domestic and farm animals, and zoo, sports or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is a human.

[0205] Thus, in some embodiments, the cell is a mammalian cell, an engineered mammalian cell or cell line, or a mammalian immune cell.

[0206] In some embodiments, the mammalian cell is an immune cell.

[0207] In some embodiments, the immune cell may be, but is not limited to, a B cell, a T cell or a hybridoma, preferably a B cell.

[0208] In some embodiments, a cell or cells herein refers to cells of different types or cells of the same type or origin exposed to different conditions.

[0209] In a specific embodiment, the cell is a non-mammalian cell.

[0210] In a further specific embodiment, the non-mammalian cell is a yeast cell, an avian cell or a shark cell.

[0211] As mentioned above, the single-cell droplet typically comprises an aqueous composition, wherein the aqueous composition is typically appropriate for the cells used and typically comprises a buffer solution as defined herein below.

[0212] As is well known to those skilled in the art, cells contain nucleic acids, wherein nucleic acids are defined as described herein above.

[0213] In the context of the method of the invention, the cells are preferably living cells. Methods for separating living cells from dead cells are well known to those skilled in the art.

[0214] Cell lysis

[0215] As is well known to those skilled in the art, nucleic acids present in single cells are released when the cells are lysed.

[0216] In the context of the present invention, "cell lysis" can be achieved by enzymatic, physical and / or chemical methods or any combination thereof, in particular enzymatic, physical and / or chemical methods. Other cell disruption methods can also be used. When present in droplets (such as single cell droplets or fusion droplets), or when present in a reservoir without droplets, the cell lysis methods described below can be used to lyse cells.

[0217] Thus, in some embodiments, single cells or cells are lysed using enzymatic, physical and / or chemical cell lysis in the lysis step in the context of the methods of the invention.

[0218] "Enzymatic methods" for removing cell walls are well-recognized in the art. Enzymes are generally commercially available and, in most cases, are initially isolated from biological sources. Commonly used enzymes include lysozymes, lysostaphins, schizolysins, mutanolysins, glycanases, proteases, and mannose.

[0219] As is well known to those skilled in the art, "chemical cell lysis" is achieved by chemical substances such as detergents, which destroy the lipid barrier around cells by destroying lipid-lipid, lipid-protein and protein-protein interactions. The ideal detergent for cell lysis depends on the cell type and source. Nonionic and zwitterionic detergents are milder detergents. Triton X series nonionic detergents and zwitterionic detergent 3-[(3-chloropropyl) dimethylamino]-1-propanesulfonate (CHAPS) are commonly used for these purposes. On the contrary, ionic detergents are strong solubilizers and tend to denature proteins, thereby destroying the activity and function of proteins. SDS is an ionic detergent that binds and denatures proteins and is widely used in the art to destroy cells.

[0220] "Physical cell lysis" refers to the use of sonication, heat shock, ice shock or electroporation.

[0221] In one example, the cells are lysed using cold shock. In the context of the method of the invention, this can be achieved by cooling a chip containing, for example, single cells in multiple reservoirs.

[0222] As described herein below, in some embodiments, an RT droplet or multiple RT droplets, a fused droplet or multiple fused droplets, or multiple reservoirs contain a lysis composition.

[0223] In some embodiments, the lytic composition comprises an enzyme selected from the group consisting of lysozyme, lysostaphin, serotonin, mutanolysin, glycanase, protease, and mannose.

[0224] In a preferred embodiment, in the context of the present invention, the lysis composition comprises magnesium chloride, a detergent, a buffer solution and an RNase inhibitor.

[0225] In 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.

[0226] Non-limiting examples of buffer solutions can include Tris-HCl, Hepes-KOH, Pipes-NaOH, maleic acid, phosphoric acid, citric acid, malic acid, formic acid, lactic acid, succinic acid, acetic acid, pivalic acid (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, H EPPS, HEPPSO, Tris, trimethylglycine, glycine, diglycine, 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). Non-limiting examples of RNase inhibitors can include RNase OUT, IN, SuperINRnase and those inhibitors targeting a wide range of RNAses (e.g., A, B, C, 1 and T1).

[0227] As described in the section "Microfluidic methods for barcoding single-cell nucleic acids" below, when applied to droplets, chemical lysis typically occurs after fusing the single-cell droplet with the RT droplet. In some embodiments, cell lysis preferably occurs after performing a phenotypic assay, such as after step b) of a method for genotyping a single cell with a phenotype of interest.

[0228] As one skilled in the art will appreciate, components of the reverse transcriptase and reverse transcriptase compositions and lysis compositions will be diluted when other components are added, for example, in one example, the lysis composition will be diluted when the RT droplets and single-cell droplets fuse.

[0229] Therefore, the concentrations given for the components of the lysis composition are preferably given as final concentrations, for example after droplet fusion.Thus, one skilled in the art will appreciate to adjust the initial concentrations present in the reservoir or RT droplets.

[0230] In some embodiments, the concentration of magnesium chloride is 1 mM to 20 mM.

[0231] In some embodiments, the concentration of the detergent is 0.1% to 10%.

[0232] In one example, the lysis composition is typically 0.2% Triton, 3 mM MgCl2, 50 mM Tris-HCl pH 7.4.

[0233] Reverse transcription (RT)

[0234] In one step in the context of the method of the invention, a plurality of RT droplets are injected, wherein at least some of the RT droplets comprise a reverse transcriptase (RT) and at least one oligonucleotide.

[0235] In the context of a microfluidic method for genotyping a single cell having a phenotype of interest, a reverse transcriptase (RT) and at least one oligonucleotide are provided in each of a plurality of reservoirs.

[0236] "Reverse transcriptase (RT)" is an enzyme used in a process known as reverse transcription to produce complementary DNA (cDNA) from a nucleic acid template, particularly an RNA template.

[0237] In 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.

[0238] In some embodiments, the reverse transcriptase has template switching activity.

[0239] Template switching activity allows the use of universal sequences to uniquely label cDNA. Using template switching activity results in unique labeling of cDNA with the universal sequence at its 5' end. In this application, universal sequence refers to a sequence that is commonly used for rapid amplification (RACE) of the 5' end of cDNA. Those skilled in the art generally know how to perform 5' RACE and which universal sequence can be used.

[0240] In the context of the method of the invention, reverse transcription is performed after fusion of at least one droplet of the first type with at least one droplet of the second type, or optionally when the method is performed in the absence of droplets, after step c) of the method for genotyping single cells having a phenotype of interest.

[0241] Thus, as described herein above in the context of lysis compositions, it will be understood by those skilled in the art that when different components (e.g., lysis compositions, reverse transcriptase or reverse transcriptase compositions) are mixed, components such as reverse transcriptase will be diluted. Thus, concentrations given for any component herein below are preferably given in terms of the final concentration present, e.g., after droplet fusion.

[0242] In some embodiments, the concentration of reverse transcriptase is 1 to 50 U / μΙ, preferably 5 to 25 U / μΙ, for example 12.5 U / μΙ.

[0243] "Reverse transcription" or "RT reaction" is a method in which single-stranded RNA is reverse transcribed into single-stranded complementary DNA (cDNA) by using total cellular RNA or poly (A) RNA, reverse transcriptase, primers, dNTPs and RNase inhibitors. It will be understood by those skilled in the art that the product of reverse transcription is an RNA / DNA duplex comprising a single-stranded cDNA hybridized to its template RNA. It will be further understood that the RNA / DNA duplex is further linked to at least one oligonucleotide comprising a primer sequence for reverse transcription.

[0244] Thus, one skilled in the art will appreciate that reverse transcribing a nucleic acid (e.g., in step b) of a microfluidic method for barcoding single-cell nucleic acid) results in the production of single-cell cDNA.

[0245] Thus, in some embodiments, at least some of the fused droplets also contain single-cell cDNA generated by reverse transcription of nucleic acid from a single-cell lysate.

[0246] Thus, in some embodiments, when droplets are not used, the reservoir also contains single-cell cDNA generated by reverse transcription of nucleic acid from a single-cell lysate.

[0247] In some embodiments, the cDNA is a single-stranded complementary DNA.

[0248] In a further embodiment, the cDNA is contained in an RNA / DNA duplex.

[0249] In some embodiments, an RNA / DNA duplex refers to RNA that has been reverse transcribed and is still partially or fully hybridized to a primer sequence for synthesizing cDNA and / or at least one polynucleotide.

[0250] As will be appreciated by those skilled in the art, in some embodiments, the RNA / DNA duplex is linked to at least one oligonucleotide comprising a primer sequence to which the nucleic acid (preferably mRNA) hybridizes during the hybridization step (e.g., in step a)) and which is used for reverse transcription during the reverse transcription step (e.g., in step b)).

[0251] As will be appreciated by one skilled in the art, in some embodiments at least one oligonucleotide, and thus the RNA / DNA duplex, is attached to a first type of particle.

[0252] In some embodiments, a plurality of reservoirs, a RT droplet or a plurality of RT droplets, or a fused droplet or a plurality of fused droplets comprises a reverse transcriptase composition.

[0253] In some embodiments, the reverse transcriptase composition comprises a protease inhibitor, dNTPs and / or DTT, preferably a protease inhibitor, dNTPs and DTT.

[0254] In some embodiments, the protease inhibitor comprises a plurality of protease inhibitors.

[0255] In some embodiments, the protease inhibitor is selected from the following list: leupeptin hemisulfate, pepstatin A, AEBSF, aprotinin, bestatin hydrochloride, E-64, and PMSF.

[0256] For example, the protease inhibitor may comprise one or more of leupeptin hemisulfate, pepstatin A, AEBSF, aprotinin, bestatin hydrochloride, E-64, and PMSF.

[0257] As used in this application, the term "dNTP" refers to a deoxynucleoside triphosphate, such as 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 also intended to refer to deoxynucleoside triphosphates including modified bases and base analogs that can mimic the base pairing of A, C, G, T, or U, or can base pair in a degenerate pattern, such as bases that pair with A or G, C or T, A or C, G or T, G or C, or A or T, referred to as nucleotide analogs. The nucleotide analogs can be used, for example, for purification, as further described herein below.

[0258] It will be appreciated by those skilled in the art that concentrations given for components of a reverse transcriptase composition are preferably given at the final concentration present in the fused droplets, for example following droplet fusion.

[0259] In some embodiments, the concentration of DTT is 1 mM to 10 mM, preferably 5 mM.

[0260] In some embodiments, the concentration of dNTPs is 0.01 mM to 10 mM, preferably 0.1 to 1 mM, more preferably 0.5 mM.

[0261] In some embodiments, the reverse transcriptase composition further comprises an RNase inhibitor. RNase inhibitors are defined herein above in the section "Cell Lysis".

[0262] Oligonucleotides

[0263] The term "oligonucleotide" is as defined herein above.

[0264] In the context of the microfluidic methods of the present invention, "at least one oligonucleotide" comprises a primer sequence.

[0265] A "primer sequence" is typically a short, single-stranded nucleic acid, between 10 and 50 nucleotides in length, designed to completely or nearly completely match a nucleic acid of interest in order to capture it and then amplify it by conventional PCR or reverse transcribe it by conventional RT. A primer sequence is "specific" for the nucleic acid to which it hybridizes, i.e., it hybridizes preferably under stringent hybridization conditions, more preferably under highly stringent hybridization conditions, or is complementary or nearly complementary to the nucleic acid to which it hybridizes (also called a target sequence).

[0266] "Stringent conditions" or "highly stringent conditions" are as defined herein above.

[0267] Typically, the primer sequence is used as the starting point for nucleic acid synthesis, allowing a polymerase such as a nucleic acid polymerase to extend the primer sequence and replicate a complementary strand. The primer sequence can be complementary to the target nucleic acid and hybridize with the target nucleic acid. In some embodiments, the primer sequence is a synthetic primer sequence.

[0268] In some embodiments, the primer sequence is a non-naturally occurring primer sequence. The primer sequence generally has a length of 10 to 50 nucleotides. For example, the primer sequence can 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, the primer sequence has a length of 18 to 24 nucleotides.

[0269] In 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.

[0270] The "poly T sequence" herein refers to a sequence containing 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 mRNA.

[0271] In some embodiments, the random DNA sequence can be 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.

[0272] In a specific embodiment, the primers are gene-specific sequences and the genes are selected from the group consisting of an antibody heavy chain variable gene, an antibody heavy chain constant gene, an antibody light chain variable gene, an antibody light chain constant gene, an αT cell receptor gene (variable or constant), a βT cell receptor gene (variable or constant), a δT-cell receptor gene (variable or constant), or a gene from a panel selected for transcriptome analysis.

[0273] The term “antibody” in the expressions “antibody heavy chain variable gene”, “antibody heavy chain constant gene”, “antibody light chain variable gene” and “antibody light chain constant gene” are as defined herein above.

[0274] The term "T cell receptor" in the expression "α T cell receptor gene", "β T cell receptor gene" and "δ T cell receptor gene" or "γ T cell receptor gene" is as defined herein above.

[0275] The term "gene" is as defined herein above.

[0276] It will be understood by those skilled in the art that the number of at least one oligonucleotide present in each reservoir of the plurality of reservoirs or in one RT droplet will be appropriate to the number of different nucleic acids (particularly the number of RNA) to be transcribed or barcoded from a single cell.

[0277] Thus, the "at least one" in the expression "at least one oligonucleotide" refers to the number of different oligonucleotides present in one droplet or reservoir, wherein the primer sequence of one oligonucleotide of the at least one oligonucleotide is different from the other oligonucleotides.

[0278] In some embodiments, the "at least one" in the phrase "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.

[0279] It should be understood that "at least one oligonucleotide" therefore refers to at least one oligonucleotide present in a certain concentration in the RT droplet or each reservoir, and that one oligonucleotide from at least one oligonucleotide therefore refers to multiple oligonucleotides having the same sequence.

[0280] It will also be understood by those skilled in the art that, for example, when the transcriptome of a cell is transcribed and barcoded, oligonucleotides having primer sequences specific for all mRNAs, such as poly T primer sequences, as will be further defined below in this application, will be used, whereas when a gene-specific transcriptome is transcribed and barcoded, oligonucleotides comprising gene-specific primer sequences are used, as will be further defined below in this document.

[0281] Thus, it will be appreciated that the number of oligonucleotides corresponds at least to the number of specific genes whose transcriptomes are to be transcribed and barcoded.

[0282] In a 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 to another gene (such as an antibody light chain variable gene).

[0283] In another 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 for, e.g., an α T cell receptor gene or a β T cell receptor gene or a γ T cell receptor gene, and wherein the other of the at least two oligonucleotides comprises a primer sequence specific for, e.g., a δ T cell receptor gene.

[0284] In another specific 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, e.g., an α T cell receptor gene and the second of the at least three oligonucleotides comprises a primer sequence specific for, e.g., a β T cell receptor gene, and wherein the third of the at least three oligonucleotides comprises a primer sequence specific for, e.g., a poly A mRNA.

[0285] "Transcriptome" generally refers to the collection of all messenger RNA molecules in a cell or a cell population. Therefore, in this application, "cell transcriptome" or "single cell transcriptome" refers to the collection of all messenger RNA molecules in a cell.

[0286] As will be appreciated by those skilled in the art, a gene-specific transcriptome therefore refers to the set of all messenger RNA molecules from a gene.

[0287] As known to those skilled in the art, different gene products, referred to as isoforms, can be encoded by a gene. Therefore, a gene-specific transcriptome can also refer to messenger RNA molecules of at least one specific isoform of a specific gene, such as messenger RNA molecules of 1, 2, 3, 4 or more specific isoforms of a specific gene or messenger RNA molecules of all isoforms of a specific gene.

[0288] Thus, in some embodiments, the single-cell nucleic acid in the context of the present invention is RNA, wherein RNA is as defined herein above.

[0289] In other embodiments, the mRNA comprises a poly A sequence, also referred to as a poly A tail.

[0290] In addition, in some specific embodiments, in the context of the present invention, "at least some nucleic acids" refers to at least one nucleic acid, preferably at least 2, at least 3, at least 4, at least 5 or more nucleic acids. In a specific embodiment, the nucleic acids of step a) and step b) refer to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleic acids.

[0291] The method of the present invention can be used to reverse transcribe from one specific nucleic acid to all nucleic acids of a single cell.

[0292] Thus, in other embodiments, in the context of the present invention, "at least some nucleic acids" refers to 1 to 100,000 nucleic acids, such as 1 to 80,000, 1 to 60,000, 1 to 40,000, such as 1, 1000, 2000, 4000, 6000, 8000, 10000, 12000, 14000, 16000, 20000, 25000, 30000, 45000, 50000 nucleic acids. Thus, in some embodiments, nucleic acids in the present application refer to the RNA of all genes.

[0293] As mentioned above, the methods of the present invention are also referred to as reverse transcriptase gene characterization transcriptomes.

[0294] Therefore, in some embodiments, the nucleic acid in the present application refers to the RNA of at least one specific gene.

[0295] The at least one specific gene can be at least 1, at least 2, at least 3, at least 4, at least 5, such as 1 to 10, 1 to 5 genes, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 genes.

[0296] In some embodiments, the at least one specific gene is selected from the group consisting of an antibody heavy chain variable gene, an antibody heavy chain constant gene, an antibody light chain variable gene, an antibody light chain constant gene, an αT cell receptor gene, a βT cell receptor gene, a δT cell receptor gene, and a γT cell receptor gene.

[0297] In some embodiments, specific genes can also be selected to further obtain cell phenotypic information and classify cells based on gene expression.

[0298] In some embodiments, at least one oligonucleotide further comprises a promoter and / or a spacer sequence.

[0299] The promoter and / or spacer sequence is preferably located at the 5' end or at the 5' end of the primer sequence. Examples of promoter sequences include, but are not limited to, T7 promoter, T3 promoter or SP6 promoter.

[0300] In some embodiments, the oligonucleotide further comprises a barcode sequence.

[0301] Thus, in some embodiments, an oligonucleotide comprises a barcode sequence and a primer sequence from 5' to 3'.

[0302] In the present application, "barcode sequence" or simply "barcode" refers to a unique nucleic acid sequence that can be distinguished from another nucleic acid sequence by its sequence, thus allowing a nucleic acid sequence to be uniquely labeled so that it can be distinguished from another nucleic acid carrying another barcode sequence.

[0303] In some embodiments, a barcode sequence uniquely identifies nucleic acid released by a single cell from nucleic acid released by other cells, for example, even after the nucleic acids are mixed together.

[0304] In some embodiments, barcode sequences are used to distinguish between dozens, hundreds, or even thousands of nucleic acids, for example, from different cells or other sources.

[0305] In some embodiments, the barcode sequence is any suitable length. A length sufficient to distinguish the barcode sequence from other barcode sequences is a preferred length of the barcode sequence. In some embodiments, the 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.

[0306] In some embodiments, a barcode sequence is composed of more than one barcode sequence, and thus can be referred to as "at least one barcode sequence."

[0307] In a related embodiment, the different barcode sequences can be from a "pool" of potential barcode sequences, which themselves are typically generated by split and poll synthesis. If the barcode sequence is composed of more than one barcode sequence, the barcode sequences can be from the same or different pools of potential barcode sequences. The pool of sequences can be selected using any suitable technique, such as randomly, or to enable error detection and / or correction of the sequences, such as being separated by a distance (e.g., a Hamming distance) to enable detection and, in some cases, correction of errors in the barcode sequence reads. The library can be any number of potential barcode sequences, such as 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.

[0308] Methods for ligating barcode sequences from one "pool" or more than one "pool" together are well known to those of skill in the art and include, but are not limited to, methods using a ligase and / or using annealing or primer extension.

[0309] Non-limiting examples of ligases include DNA ligases, such as DNA ligase I, DNA ligase Ligase II, DNA ligase III, DNA ligase IV, T4 DNA ligase, T7 DNA ligase, T3 DNA ligase, E. coli DNA ligase, Taq DNA ligase, etc. Many such ligases are commercially available.

[0310] In some embodiments, the barcode sequence is a double-stranded or single-stranded nucleic acid.

[0311] In some embodiments, the oligonucleotide further comprises a unique molecular identifier (UMI). The UMI is located at the 3' end or the 5' end of the barcode sequence.

[0312] "Unique molecular identifier" (UMI) sequences are well known to those skilled in the art and are described, for example, in Kivioja et al. (Nature Method, 2012, vol 9, N°1). UMI sequences allow for the biological information removal of duplicate reads.

[0313] In some embodiments, at least one oligonucleotide is introduced into a reservoir or a droplet, in particular into a RT droplet, by initially binding the at least one oligonucleotide to at least one particle of a first type.

[0314] It will be appreciated by those skilled in the art that temporarily binding at least one oligonucleotide to a particle allows providing particles with a large number of oligonucleotides. In addition, initially binding at least one oligonucleotide to a particle facilitates the introduction of at least one oligonucleotide in each droplet (such as RT droplets), particularly when droplets are used.

[0315] Thus, in some embodiments, the reservoir or RT droplet further comprises at least one first type of particle associated with at least one oligonucleotide.

[0316] At least one first type of particle in the present application refers to at least 1, 2, 3, 4, 5 or more first type of particles, preferably one first type of particle.

[0317] As will be understood by those skilled in the art and as explained above, the number of particles of the first type encapsulated in one RT droplet and the size of the droplet follow a probability distribution, such as a Poisson distribution, and depend on the concentration of the first type of particles, the injection parameters of the aqueous composition (such as a reverse transcription composition or a lysis composition), the geometry of the main channel, and the injection parameters of the first type of particles and the carrier fluid.

[0318] Thus, in some embodiments, parameters can be adjusted to obtain RT droplets having 2, 1, or 0 particles of the first type therein, thereby avoiding droplets containing more than 2 particles of the first type.

[0319] In a related embodiment, parameters may be adjusted to obtain RT droplets having 1 or 0 particles of the first type therein, thereby avoiding droplets containing more than 1 particle of the first type.

[0320] In some embodiments, at least one first type of particle is encapsulated in the RT droplet, preferably, each droplet contains no more than about 2 first type particles. In other embodiments, at least one first type of particle is encapsulated in the droplet at no more than about 1 particle / droplet, or at least one first type of particle is preferably encapsulated in the droplet at 1 particle / droplet, or at least one first type of particle is encapsulated in the droplet at an average of 1 particle / droplet.

[0321] Similarly, when droplets are not used, in some embodiments, each reservoir preferably contains no more than about 2 particles of the first type per reservoir, preferably no more than about 1 particle per reservoir, or 1 particle per reservoir, or an average of 1 particle per reservoir.

[0322] In the context of the present invention, "particles", such as first type of particles or second type of particles, refer to microparticles.

[0323] In some embodiments, the particle is a hydrogel particle, a polymer particle, or a magnetic particle.

[0324] The particles may have an irregular or regular shape. For example, the particles may be spherical, ellipsoidal or cubic.

[0325] In some embodiments, in the context of the present invention, the particles are hydrogel particles.

[0326] For example, "hydrogel particles" are described in International Patent Application No. WO 2008 / 109176 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 bisacrylamide, polyacrylamide, streptavidin acrylamide, poly-N-isopropylacrylamide, or poly-N-isopropylacrylamide, or mixtures thereof. In one example, the hydrogel particles include acrylamide, bisacrylamide, and streptavidin acrylamide.

[0327] In another set of embodiments, the particles comprise one or more polymers and are therefore referred to herein as "polymer particles." 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 copolymers of these and / or other polymers.

[0328] Furthermore, in some embodiments, the particles are magnetic, and are therefore referred to as "magnetic particles," which can allow magnetic manipulation of the particles.

[0329] For example, the magnetic particles may contain iron or other magnetic substances.

[0330] The particles (particularly the first or second type of particles) can also be functionalized so that other molecules, such as proteins, nucleic acids or small molecules, can be attached to them. Thus, some embodiments of the present invention relate to a set of particles, which are defined as a library of, for example, nucleic acids, proteins, small molecules or other substances (such as those described in the present application). In one example, the particles are functionalized using antibodies. In some embodiments, the antibodies are directed against components of cells.

[0331] In some embodiments, the particles (particularly the first type of particles) are fluorescent.

[0332] In some embodiments, the particle comprises streptavidin.Streptavidin may be coupled to the surface of the particle as defined herein above.

[0333] In some embodiments, the size of the first or second particle is 0.1 pL to 100 pL, such as 0.1 pL to 500 pL, 0.1 pL to 400 pL, 0.1 pL to 400 pL, 0.1 pL to 300 pL, for example 0.5 pL to 300 pL, 0.5 pL to 250 pL, 0.5 pL to 200 pL, 1 pL to 250 pL, 1 pL to 200 pL, preferably 1 pL to 200 pL, typically 100 pL to 200 pL, such as 150 pL.

[0334] In some embodiments, at least one oligonucleotide is covalently or non-covalently linked to at least one first type of particle, wherein at least one first type of particle is as defined herein above.

[0335] In this context, "non-covalent bonding" refers to, for example, a streptavidin-biotin bond. Other non-covalent bonds are well known to those skilled in the art, such as avidin-biotin bonds or his tag and nickel bonds thereof.

[0336] In the present application "covalently linked" refers to, for example, an amino bond or an acrylic phosphoramidite bond.

[0337] "Streptavidin" usually refers to a 52.8 kDa protein purified from the bacterium Streptomyces avidinii. Streptavidin homotetramers have a very high affinity for biotin, with a dissociation constant (Kd) of the order of ≈10 - 14 mol / L, the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature.

[0338] In a preferred embodiment, the non-covalent bond is a streptavidin-biotin linkage.

[0339] Streptavidin-biotin bonds are well known to those skilled in the art.

[0340] Thus, in some embodiments, the first type of particle comprises, for example, streptavidin, and in the same embodiment, at least one oligonucleotide comprises biotin. In other words, at least one type of oligonucleotide is biotin functionalized.

[0341] Independently of the type of bond used to connect the at least one type of oligonucleotide to the first type of particle, the at least one type of oligonucleotide may further comprise at least one linker sequence.

[0342] Therefore, in other embodiments, "at least one type of oligonucleotide" or simply "oligonucleotide" further comprises at least one linker sequence, which is preferably included near the 5' end.

[0343] In some cases, in addition to the primer sequences described above, the oligonucleotides may also contain one or more primers for sequencing. Examples of such primer sequences for sequencing include, but are not limited to, P5 primers, P7 primers, PE1 primers, PE2 primers, A19 primers, or other primers discussed herein.

[0344] Thus, in some embodiments, at least one type of oligonucleotide comprises from 5' to 3' optionally an adapter sequence, optionally a promoter sequence, optionally a primer sequence for sequencing, optionally a barcode sequence, optionally a UMI, and a primer sequence.

[0345] Thus, those skilled in the art will appreciate that in some embodiments, at least one oligonucleotide is bound to the first type of particle via the 5' end.

[0346] However, in some alternative embodiments, at least one oligonucleotide may be bound to the first type of particle via the 3' end.

[0347] In some embodiments, a "linker sequence" is a sequence by which at least one oligonucleotide is optionally linked to a first type of particle.

[0348] "Optionally attached herein" refers to the possibility that once at least one type of oligonucleotide attached to a particle is loaded into an RT droplet or multiple RT droplets or reservoirs, the at least one type of oligonucleotide can be released so that the RT droplet or fused droplet or reservoir contains a first type of particle and at least one oligonucleotide, while the at least one type of oligonucleotide is not attached to the first type of particle.

[0349] Preferably, the linker sequence is a cleavable linker sequence, for example capable of being cleaved upon application of a suitable stimulus, such as enzymatic and / or photocleavage.

[0350] "Cleavable linkers" may include, but are not limited to, TEV, trypsin, thrombin, cathepsin B, cathepsin D, cathepsin K, caspase lumatrix metalloproteinase sequences, phosphodiesters, phospholipids, esters, -galactose, dialkyldialkoxysilanes, cyanoethyl groups, sulfones, ethylene glycol disuccinates, 2-N-acylnitrobenzenesulfonamides, α-thiophenyl esters, unsaturated vinyl sulfides, activated sulfonamides, malondialdehyde (MDA)-indole derivatives, levulinic acyl esters, hydrazones, acylhydrazones, alkylthioesters, disulfide bridges, azo compounds, 2-nitrobenzyl derivatives, benzoyl esters, 8-quinolylbenzenesulfonate, coumarins , phosphotriesters, bisarylhydrazones, bimane dithiopropionic acid derivatives, p-methoxybenzyl derivatives, tert-butylcarbamate analogs, dialkyl or diaryldialkoxysilanes, orthoesters, acetals, aconityl, hydrazones, β-thiopropionic acid esters, phosphoramidates, imines, trityl, vinyl ethers, polyketals, alkyl 2-(diphenylphosphino)benzoate derivatives, allyl esters, 8-hydroxyquinoline esters, picolinates, vicinal diols, and selenium compounds (see, e.g., Leriche G, Chisholm L, Wagner A).

[0351] Cleavable linkers are well known to those skilled in the art and are further described in chemical biology, for example, Leriche H. et al. (Bioorg Med Chern. 15; 20(2): 571-82. 2012). Cleavage conditions and reagents include, but are not limited to, enzymes, nucleophilic / alkaline reagents, reducing agents, photoirradiation, electrophilic / acidic reagents, organometallic and metal reagents, and oxidizing agents.

[0352] In some embodiments, the method of the invention further comprises the step of releasing at least one oligonucleotide bound to at least one first type of particle from said particle, for example after incorporating it into a RT droplet or after providing it in a reservoir.

[0353] Furthermore, in some embodiments, prior to reverse transcription of the single-cell nucleic acid, at least one oligonucleotide initially bound to at least one first type of particle is released from the at least one first type of particle.

[0354] In some embodiments, the step of releasing at least one oligonucleotide can occur before or after fusing the single cell with the RT droplet, after lysing the cell, and / or before reverse transcribing the nucleic acid of the single cell.

[0355] It will be understood by the skilled person that, depending on the time point chosen for releasing at least some of the oligonucleotides, the term "at least some of the oligonucleotides" may refer, for example, to at least some of the oligonucleotides hybridized to nucleic acids or DNA / RNA duplexes released by the cell, as defined herein above.

[0356] In some embodiments, any method can be used to release at least some of the oligonucleotides, such as enzymes, nucleophilic / basic reagents, reducing agents, light radiation, electrophilic / acidic reagents, organometallic and metallic reagents, and oxidizing agents.

[0357] In some embodiments, enzymatic and / or photocleavage can be used to release at least one oligonucleotide. For example, an endonuclease can be used to cleave a linker sequence or any other sequence to release at least some oligonucleotides from the first type of particle.

[0358] In other embodiments, releasing the oligonucleotide refers to breaking a bond, such as a streptavidin-biotin bond. Methods for breaking a streptavidin-biotin bond are well known to those skilled in the art and include enzymatic digestion of streptavidin and / or denaturation of streptavidin.

[0359] In some embodiments, the oligonucleotide is released by enzymatic digestion of streptavidin.

[0360] The concentration of the at least one oligonucleotide is 10 nM, preferably at least 100 nM.

[0361] In some embodiments, the concentration of at least one oligonucleotide is at least 150 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM and at least 1 μM, such as 100 nM to 5 μM, 100 nM to 4 μM, 100 nM to 3 μM, 100 nM to 2 μM, 100 nM to 1 μM, preferably 100 nM to 500 nM.

[0362] In one example, the primer sequence is a poly T primer sequence and the concentration of the at least one oligonucleotide is 100 nM to 3300 nM (corresponding to 3.3 μΜ).

[0363] In other examples, the primer sequence is a gene-specific primer sequence and the concentration of the at least one oligonucleotide is 100 nM or 1000 nM (corresponding to 1 μΜ).

[0364] In some embodiments, the concentration of each oligonucleotide in the at least one oligonucleotide in the microfluidic droplet is at least 10 nM, preferably at least 100 nM.

[0365] In some embodiments, the concentration of each oligonucleotide in 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 1μΜ, such as 100nM to 5μΜ, 100nM to 4μΜ, 100nM to 3μΜ, 100nM to 2μΜ, 100nM to 1μΜ, preferably 100nM to 500nM.

[0366] According to the above, in some embodiments, the reservoir, RT droplet and the plurality of RT droplets further comprise a lysis composition, wherein the lysis composition is defined in the section "Cell Lysis" herein above.

[0367] In some embodiments, the concentration of at least one oligonucleotide refers to the concentration in the reservoir, in the RT droplet, or in the fusion droplet.

[0368] In some embodiments, the RT droplets can be screened and sorted prior to injecting the plurality of RT droplets.

[0369] Thus, in some embodiments, the RT droplets further comprise a second type of particle, wherein the second type of particle is as defined in the section "Single Cells".

[0370] Therefore, in some other embodiments, the RT droplets further comprise a dye as defined herein above.

[0371] Amplification and sequencing

[0372] In some embodiments, the method of the present invention further comprises a step of amplifying the barcoded single cell cDNA after recovering the barcoded single cell cDNA. In some embodiments, the amplification step is performed after removing unincorporated oligonucleotides. In some embodiments, the amplification step is performed before a sequencing step as defined herein below.

[0373] In some embodiments, the amplification step is performed in a multiplex reaction, a separate polymerase chain reaction (PCR) (see Fig.11 ), isothermal amplification or linear amplification.

[0374] In some embodiments, the linear amplification is in vitro transcription.

[0375] In some embodiments, the barcoded single-cell cDNA produced in the methods of the invention is quantified using qPCR (eg, single and / or multiplex qPCR reactions).

[0376] In other embodiments, the method of the present invention further comprises the step of sequencing the barcoded single-cell cDNA.

[0377] In the context of the present invention, in some embodiments, the steps of sequencing the barcoded single-cell cDNA described herein refer to first contacting the barcoded single-cell cDNA with a sequencing library, and amplifying sequences of interest from the sequencing libraries corresponding to the barcoded single-cell cDNA, respectively.

[0378] In some embodiments, the step of sequencing the barcoded single cell cDNA may include performing a next generation sequencing (NGS) protocol on the sequencing library (see Fig.12 )

[0379] In certain embodiments, the NGS protocol includes loading the sequencing library in an amount between 4 pM and 20 pM per flow cell of the kit.

[0380] In some embodiments, the NGS sequencing protocol further comprises the step of adding 5-60% PhiX to a certain amount of sequencing library or to a flow cell of the kit.

[0381] Microfluidic methods for barcoding single-cell nucleic acids

[0382] "Barcoding" herein refers to the addition of a gene sequence, called a barcode sequence, as defined herein above in the section "Oligonucleotides" to a nucleic acid, which is capable of distinguishing the barcoded nucleic acid from a nucleic acid having another added gene sequence (ie, another unique barcode sequence).

[0383] The inventors have developed a method (see Figure 8 ), wherein a single cell droplet containing nucleic acid is specifically fused with a droplet containing reverse transcriptase and oligonucleotides with primer sequences in a microfluidic device comprising a chip. Then, during reverse transcription or thereafter, the nucleic acid is reverse transcribed and ultimately barcoded. One advantage of this method is that only a small amount of material is wasted because a single cell droplet is specifically fused with a RT droplet. Another advantage is that unused barcodes are removed, so when cDNA sequencing is performed, the signal-to-noise ratio is improved.

[0384] Another advantage is that the barcodes themselves can be used as primers to continue single-cell RT-PCR (see Figure 7 ).

[0385] Therefore, the present invention relates to a microfluidic method for barcoding single-cell nucleic acids, the method comprising:

[0386] - providing a microfluidic device comprising a chip, the chip comprising at least one microfluidic channel and a plurality of reservoirs,

[0387] - injecting a carrier fluid into an inlet of the microfluidic channel, the carrier fluid comprising a plurality of first type droplets dispersed in the carrier fluid, wherein the first type droplets are 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 a single cell, wherein the single cell comprises a single-cell nucleic acid,

[0388] - for a plurality of reservoirs, a first migration step, wherein at least one droplet of the first type in the plurality of droplets is moved into a reservoir of the plurality of reservoirs by buoyancy,

[0389] - injecting a carrier fluid into an inlet of the microfluidic channel, the carrier fluid comprising a plurality of droplets of a second type dispersed in the carrier fluid, wherein the droplets of the second type are 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,

[0390] - for a plurality of reservoirs, a second migration step, wherein at least a portion of at least one droplet of the second type enters a reservoir of the plurality of reservoirs,

[0391] - for a plurality of reservoirs, said at least one droplet of said first type merges with said at least one droplet of said second type in each reservoir or at its edge, thereby producing a fused droplet,

[0392] - and further comprising the following steps:

[0393] a) for each fused droplet, hybridizing at least some of the single-cell nucleic acid to the at least one oligonucleotide in the fused droplet,

[0394] b) in each fused droplet, reverse transcribing at least some of the single-cell nucleic acid present in the fused droplet, thereby producing single-cell cDNA, and

[0395] c) attaching at least one barcode sequence to the single cell cDNA obtained in step b), wherein the at least one barcode sequence encodes the identity of the single cell,

[0396] or

[0397] - It also includes the following steps:

[0398] a) for each fused droplet, hybridizing at least some of the single-cell nucleic acid from at least one cell to the at least one oligonucleotide in the fused droplet, wherein the at least one oligonucleotide comprises at least one barcode sequence,

[0399] b) in each fused droplet, reverse transcribing at least some of the single-cell nucleic acids present in the fused droplet, thereby generating barcoded single-cell cDNA, wherein the at least one barcode sequence encodes the identity of the single cell.

[0400] Techniques for preparing and injecting single-cell droplets or RT droplets are well known to those skilled in the art and are further described in the section "Preparation of Droplets".

[0401] In some embodiments, the first type of droplets are single-cell droplets. In another set of embodiments, the first type of droplets are RT droplets. Features related to single-cell droplets and RT droplets are described in the previous sections above in this article, especially the "Droplets", "Single Cells", "Reverse Transcription" and "Oligonucleotides" sections. Therefore, the descriptions of "single-cell droplets" or "RT droplets" mentioned in these sections are fully applicable to this section entitled "Microfluidic Methods for Barcoding Single-Cell Nucleic Acids".

[0402] In the context of microfluidic methods, the "at least one oligonucleotide" comprises a primer sequence. The at least one oligonucleotide and the primer sequence are defined herein above in the section "Oligonucleotides".

[0403] In some embodiments, in the first migration step, "at least one droplet of the first type" refers to 1, 2 or 3, preferably 1 or 2, more preferably 1 droplet of the first type.

[0404] Therefore, in a preferred embodiment, in the first migration step, for a plurality of reservoirs, one droplet of the first type is received in each reservoir by buoyancy.

[0405] "Buoyancy" generally refers to the upward force exerted by a fluid opposing the weight of an immersed object. In the context of the present invention, buoyancy refers to the upward force exerted by a carrier fluid opposing the weight of a droplet immersed in the carrier fluid.

[0406] Thus, in some embodiments, the microfluidic device is oriented so that a plurality of reservoirs are located above the channel, and at least one first type of droplet enters the oriented reservoir by buoyancy. In this embodiment, the carrier fluid has a density greater than the density of the aqueous solution.

[0407] In some embodiments, the first migration step of at least one first type of droplet results in an occupation of 50% to 100% of the plurality of reservoirs, e.g., 60% to 100%, 70% to 100%, 90% to 100%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%, preferably the migration step of at least one droplet results in an occupation of 90% to 100% of the plurality of reservoirs.

[0408] "Occupancy" in this context refers to the number of reservoirs that are occupied by at least one droplet compared to the total number of reservoirs. For example, 50% occupation means that 50% of the reservoirs are occupied by at least one droplet.

[0409] In some embodiments, the first migration step of at least one first type of droplets results in a capture efficiency of the injected droplets of 10% to 100%, such as 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, preferably 80% to 100%.

[0410] "Capture efficiency" refers to the number of droplets captured in the reservoir compared to the total number of droplets injected into the inlet. For example, a capture efficiency of 50% means that the droplets captured in the reservoir account for 50% of the total number of droplets provided.

[0411] In some embodiments, in the second migration step, 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.

[0412] In the second migration step, at least one droplet of the second type may migrate completely into the reservoir or only partially, depending on the size of the reservoir, the size of the second type of droplet and the size of the second type of droplet. Those sizes are further defined in the sections "Droplets" and "Microfluidic Devices".

[0413] Thus, in some embodiments, at least a portion of at least one second type of droplets refers to 50% to 100%, e.g., 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 at least one second type of droplets.

[0414] Thus, in alternative embodiments, at least a portion of at least one second type of droplets refers to 1 to 50%, 1% to 40%, 1% to 30%, for example 2% to 30%, 5% to 30%, 10% to 30%, preferably 10% to 30%, of the total volume of at least one second type of droplets.

[0415] In some embodiments, for a plurality of reservoirs, in the second migration step, at least a portion of at least one second type of droplets enters one of the plurality of reservoirs due to buoyancy or a difference in surface energy (preferably a difference in surface energy).

[0416] In some embodiments, the second migration step of at least one second type of droplets results in 50 to 100% occupancy of the plurality of reservoirs, such as 60 to 100%, 70 to 100%, 90 to 100%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%, preferably the migration step of at least one second type of droplets results in 90 to 100% occupancy of the plurality of reservoirs.

[0417] In some embodiments, the second migration step of at least one second type of droplets results in a capture efficiency of the injected droplets of 10% to 100%, such as 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, preferably 80% to 100%.

[0418] "Occupancy" and "capture efficiency" are as defined herein above.

[0419] In some embodiments, the first type of droplets or the second type of droplets may also contain at least one second type of particle, wherein the at least one second type of particle is as defined in the section "Single Cells" above herein and the particle is as defined in the section "Oligonucleotides" above herein.

[0420] In some embodiments, for a plurality of reservoirs, the method further comprises the step of performing a phenotypic assay on the at least one single cell for each reservoir.

[0421] As will be appreciated by those skilled in the art, in some embodiments, performing a phenotypic assay may also require exposing the cell to an agent. Thus, in some embodiments, a single cell is exposed to one or more agents before, during, or after the parameter is measured.

[0422] Thus, in some embodiments, the single-cell droplet further comprises one or more reagents required to perform the single-cell assay.

[0423] Thus, in some embodiments, one or more reagents are integrated into a single-cell droplet.

[0424] In some embodiments, the phenotypic determination is performed before the step of fusing the droplets. In some embodiments, the phenotypic determination is performed before the cells are lysed. The phenotypic determination may also include the step of taking an image, thereby mapping the phenotype of the at least one single cell for each reservoir. The "phenotypic determination" and "taking an image" steps are as defined in the section "Methods for Genotyping Single Cells Having a Phenotype of Interest" below.

[0425] In the context of the methods of the present invention, the fusion step is performed on a plurality of reservoirs. As will be appreciated by those skilled in the art, fusion can be performed, for example, in selected areas of a chip, particularly if an electric field is used to fuse droplets. Thus, in some embodiments related to the fusion step, the plurality of reservoirs may refer to some of the plurality of reservoirs of a chip.

[0426] As will be appreciated by one skilled in the art, in one example, based on the results of the phenotypic assay, one skilled in the art can select multiple reservoirs in which the RT droplets and single-cell droplets will be fused.

[0427] In some embodiments, in the fusion step, for each reservoir, at least one droplet of the first type is fused with at least one droplet of the second type using temperature, electric field, demulsifier or ionic force, preferably electric field. Methods for fusing droplets are well known to those skilled in the art and are described in, for example, WO 2010128157 A1.

[0428] In some embodiments, at least one droplet of the first type is fused with at least one droplet of the second type using an electric field, wherein the electric field is 10 kHz and 500 V for at least 1 second, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10 seconds, typically 8 seconds.

[0429] In some embodiments, the fusing step results in a fusion efficiency of between 80% and 100% between the first type of droplets and the second type of droplets, such as between 85% and 100%, 90% and 100%, 92% and 100%, 93% and 100%, 94% and 100%, 95% and 100%, 96% and 100%, 97% and 100%, 98% and 100%, such as 98%, 99%, 100%, preferably 90% to 100% between the first type of droplets and the second type of droplets.

[0430] In the present application, "fusion efficiency between droplets of the first type and droplets of the second type" refers to the number of events in which fused droplets are produced by providing one droplet of the first type and one droplet of the second type compared to the total number of events in which one droplet of the first type and the second type are provided together.

[0431] In some embodiments, for multiple reservoirs, the method further comprises a step of lysing cells in, for example, at least one single-cell droplet or fused droplet to release nucleic acids from the single cells into the single-cell droplet or fused droplet.

[0432] Thus, in some embodiments of the methods of the invention, nucleic acid refers to nucleic acid released in the hybridization step (eg in hybridization step a)) and in the reverse transcription step (eg in reverse transcription step b)).

[0433] The step of lysing the cells may occur before the step of fusing at least one droplet of the first type with the at least one droplet of the second type, or after the step of fusing the droplets. Methods that may be used to lyse cells are defined herein above in the section "Cell Lysis".

[0434] Those skilled in the art will appreciate that the method chosen to lyse the cells may depend on the time point chosen for lysis, ie, lysis before or after droplet fusion.

[0435] For example, enzymatic or chemical cleavage is often used after the fusion step because the components required for enzymatic or chemical cleavage are often contained in the RT droplets.

[0436] In a preferred embodiment, cell lysis does not destroy the droplets used in the context of the present invention.

[0437] Thus, in some embodiments, the RT droplet or plurality of RT droplets comprises a lysis composition, wherein the lysis composition is defined herein above in the section "Cell Lysis".

[0438] In some embodiments, the methods of the present invention further comprise at least one clearing step.

[0439] In the context of the present invention, "at least one cleaning step" means at least 1, at least 2, at least 3, at least 4 cleaning steps.

[0440] For example, the device may typically be purged to remove entrapped air prior to injecting the plurality of droplets of the first type.

[0441] For example, the device may generally 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.

[0442] For example, the device may generally be purged after injecting the plurality of droplets of the second type to remove at least a portion of the droplets of the second type that did not migrate into the plurality of reservoirs.

[0443] For each fused droplet, step a) of the method of the invention involves hybridizing at least some of the single-cell nucleic acids in the fused droplet with at least one oligonucleotide.

[0444] In the present application, "hybridization" refers to the phenomenon of annealing of a primer sequence (e.g., a primer sequence in at least one oligonucleotide) to a complementary nucleic acid sequence in a nucleic acid. Therefore, as is well known to those skilled in the art, the temperature used for hybridization depends on the primer sequence and / or RT enzyme used.

[0445] In one example, the hybridization step (eg, hybridization step a) or b)) is performed by incubating the droplets, for example, at 55° C. to 60° C. for 1 h.

[0446] The reverse transcription of at least some of the single-cell nucleic acids in step b) is further defined herein above in the section "Reverse transcription".

[0447] In some embodiments, at least one oligonucleotide comprises a primer sequence and at least one barcode sequence, wherein "at least one oligonucleotide", "at least one barcode sequence" and "primer sequence" are as defined herein above in the section "Oligonucleotides".

[0448] In a related embodiment, thus, for example, the reverse transcription in step b) results in the production of a barcoded cDNA. According to various embodiments described herein above, the barcoded cDNA may or may not be bound to the first type of particle.

[0449] In some embodiments, the method of the present invention further comprises a step of recovering the fused droplets at the outlet of the channel after the reverse transcription step.

[0450] In this application, "recovery" refers to the collection of microfluidic droplets containing cDNA or barcoded cDNA generated by reverse transcription.

[0451] In some embodiments, recovering the fused droplets comprises the steps of rotating the device about the X-axis by 45° to 135° (preferably 90°) and clearing the device. The fused droplets can then be collected at the outlet.

[0452] In some embodiments, the step of recovering the fused droplets at the channel outlet after the reverse transcription step comprises at least one washing step.

[0453] After washing the fused droplets, downstream processing of the fused droplets can be different for droplets containing barcoded cDNA compared to droplets containing non-barcoded cDNA.

[0454] One skilled in the art will appreciate that the treatment of barcoded cDNA can be different, as barcoded single-cell cDNA obtained from one cell can be mixed with barcoded single-cell cDNA from another cell, and the cDNAs can be distinguished based on the barcode sequence, whereas non-barcoded cDNA is merely mixed when bound to particles.

[0455] Thus, in some embodiments, the cDNA is not barcoded, and in related embodiments, the cDNA produced by reverse transcription in step b) is recovered and then at least one barcode is attached in step c). After the barcode is attached in step c), the barcoded cDNA is typically used for subsequent amplification and preparation of a sequencing library (see Fig.11 ).

[0456] In some embodiments, the cDNA is barcoded, and in related embodiments, the barcoded cDNA produced by reverse transcription in step b) is recovered and typically used for subsequent amplification and preparation of a sequencing library.

[0457] Subsequent amplification and preparation of sequencing libraries are defined as above in the section "Amplification and Sequencing" (see Fig.11 ).

[0458] Therefore, in some embodiments, the methods of the present invention further comprise recovering the barcoded single-cell cDNA generated by reverse transcription in at least some of the fused droplets.

[0459] More specifically, in related embodiments, recovering refers to collecting microfluidic droplets containing barcoded cDNA generated by reverse transcription, disrupting the microfluidic droplets and separating the barcoded cDNA contained in the aqueous composition from the oil phase of the microfluidic droplets.

[0460] Methods for isolating nucleic acids (particularly barcoded cDNA) from droplets are well known to those skilled in the art and include, for example, collecting microfluidic droplets and disrupting them, typically using perfluorooctanol (v / v emulsion). Subsequently, the emulsion obtained in the previous step is incubated until the aqueous and oil phases are separated. In one example, the aqueous phase is typically centrifuged at 10,000 g for 10 min at, for example, 4°C, and the supernatant containing the cDNA is recovered.

[0461] In some embodiments, the method further comprises the step of removing unincorporated oligonucleotides.

[0462] "Removing unincorporated oligonucleotides" includes contacting an aqueous composition containing cDNA and ultimately unincorporated oligonucleotides with a purification matrix, wherein the purification matrix removes unincorporated oligonucleotides. As will be appreciated by those skilled in the art, the cDNA entering the step of removing unincorporated oligonucleotides is preferably a barcoded cDNA.

[0463] In some embodiments, the purification matrix comprises beads or particles, which are optionally formed into a column. In other examples, unincorporated oligonucleotides are removed by size selection using, for example, acrylamide gels.

[0464] In some embodiments, the step of "removing unincorporated oligonucleotides" comprises contacting the aqueous composition comprising the cDNA with an exonuclease to degrade unincorporated oligonucleotides in the aqueous composition of at least some of the droplets.

[0465] In a related embodiment, the exonuclease degrades single-stranded nucleic acid sequences in the aqueous composition comprising cDNA.

[0466] It will be appreciated by those skilled in the art that the cDNA obtained in, for example, step b) or c) (such as barcoded cDNA) is usually present in the form of an RNA / DNA complex and is therefore protected from the exonuclease.

[0467] In some embodiments, the cDNA comprises one or more nucleotide analogs as defined herein above, which facilitates purification of the cDNA sequence or molecule.

[0468] As will be appreciated by those of skill in the art, in certain embodiments, the purified cDNA does not contain unincorporated oligonucleotides.

[0469] In some embodiments, the cDNA is further treated with RNAse A.

[0470] "RNAse A" is an endoribonuclease that specifically degrades single-stranded RNA at C and U residues.

[0471] In some embodiments, the concentration of RNAse A is 10 to 1000 μg / μL, preferably 50 to 200 μg / μL, such as 100 μg / μL.

[0472] In some embodiments, the cDNA is further treated with proteinase K.

[0473] "Proteinase K" is a broad-spectrum serine protease and preferentially digests proteins after hydrophobic amino acids.

[0474] In some embodiments, the concentration of proteinase K is 0.1 to 5 mg / mL, preferably 0.1 to 1 mg / mL, such as 0.8 mg / mL.

[0475] The recovered cDNA is then usually used for subsequent amplification and preparation of a sequencing library as defined herein above in the section "Amplification and sequencing" (see also Figure 5 ).

[0476] As explained in the section “Oligonucleotides” herein above, in embodiments in which at least one oligonucleotide is bound to at least one first type of particle, reverse transcription of the nucleic acids present in the fused droplets results in single-cell cDNA being bound to at least one first type of particle in the form of an RNA / DNA complex.

[0477] Furthermore, in embodiments wherein at least one oligonucleotide does not contain a barcode sequence, the cDNA obtained by reverse transcription and bound to the at least one first type of particle is not barcoded.

[0478] Thus, in a related embodiment, recovering single-cell cDNA in the present application refers to disrupting a microfluidic droplet and separating the single-cell cDNA contained in the aqueous composition bound to at least one first type of particle from the oil phase of the microfluidic droplet.

[0479] Methods of disrupting microfluidic droplets are well known to those skilled in the art and typically involve the use of perfluorooctanol (v / v emulsion).

[0480] The cDNA bound to the particles can be separated from the oil phase using methods known to those skilled in the art. For example, when the particles are magnetic particles, a magnet can be used to separate the cDNA. In another example, when the particles are hydrogels, the cDNA can be separated by sedimentation.

[0481] Separating the cDNA bound to at least one first type of particle from the oil phase results in a library comprising a plurality of first type particles, wherein each particle is bound to the cDNA of one cell.

[0482] In a related embodiment, for example, in step c), for attaching at least one barcode sequence to the single-cell cDNA, the method of the present invention further comprises the step of dispersing the single-cell cDNA bound to at least one first type of particle into a plurality of reservoirs, wherein each reservoir receives at least one first type of particle (preferably one first type of particle) bound to the single-cell cDNA. Such a reservoir can be, for example, a well of a well plate, such as a 96-well plate.

[0483] In some related embodiments, the method of the present invention further comprises step c) attaching a barcode sequence to the single cell cDNA obtained in step b), wherein the at least one barcode sequence encodes the identity of the single cell.

[0484] At least one barcode sequence is as defined in the section "Oligonucleotides" above herein. In a preferred embodiment, when at least one barcode is attached after the transcription step, the at least one barcode sequence is preferably a single-stranded nucleic acid. It will be appreciated by those skilled in the art that one barcode sequence can be attached, or more than one barcode sequence. When more than one barcode sequence is attached, the individual barcode sequences connected to each other ultimately form one final barcode sequence. Those individual barcode sequences can be attached in subsequent cycles.

[0485] In some implementations, step c) of attaching a barcode sequence further comprises attaching a UMI sequence, wherein the UMI sequence is as defined in the section “Oligonucleotides” herein above.

[0486] Methods of attaching a barcode sequence or a UMI sequence are well known to those of skill in the art and include, for example, the use of a ligase and / or the use of annealing or primer extension methods.

[0487] In some embodiments, step c) of attaching a barcode sequence may be followed by a washing step to remove barcodes that are not attached to the single cell cDNA.

[0488] In some embodiments, the method further comprises the step of removing unincorporated oligonucleotides. The step of removing unincorporated oligonucleotides is further discussed herein above.

[0489] In embodiments where the cDNA is attached to at least one first type of particle, the method further comprises the step of releasing the cDNA or barcoded cDNA from the at least one first type of particle. Methods for releasing oligonucleotides are described in the section "Oligonucleotides" herein above, and their application is similar to the release of cDNA, since the cDNA is bound to the particle via the oligonucleotide.

[0490] Preferably, the step of releasing the cDNA occurs after attaching the barcode sequence in step c).

[0491] In some embodiments, step c) of attaching a barcode sequence is followed by a step of recovering the barcoded cDNA.

[0492] Recovering barcoded cDNA in the present application refers to collecting barcoded cDNA.

[0493] The recovered barcoded cDNA is then typically used for subsequent amplification and preparation of a sequencing library, as defined in the section "Amplification and Sequencing" herein above (see Fig.11 ).

[0494] Furthermore, in some embodiments, at least one oligonucleotide is introduced into the RT droplet without the particle, or optionally, is released from the particle prior to reverse transcription.

[0495] Thus, those skilled in the art will appreciate that, following transcription, these embodiments of the methods of the invention will result in the cDNA no longer being attached to the particle. In addition, in embodiments in which at least one oligonucleotide does not comprise a barcode sequence, the cDNA is not barcoded.

[0496] Thus, in a related embodiment, the RT droplets further comprise a polymer.

[0497] Those skilled in the art will appreciate that, therefore, in some embodiments, the fused droplets comprise the polymer.

[0498] In some embodiments, the polymer is in solution.

[0499] In some embodiments, the polymer is selected from the group consisting of alginate, agarose, poly(ethylene glycol) diacrylate or acrylamide-based gels, such as bisacrylamide, polyacrylamide, streptavidin acrylamide, poly-N-isopropylacrylamide or poly-N-isopropylacrylamide or a mixture thereof.

[0500] In other embodiments, the polymer is functionalized to capture nucleic acids.

[0501] Methods of functionalizing polymers to capture nucleic acids are well known to those skilled in the art and include, but are not limited to, streptavidin, histidine tag, biotin, calmodulin, SNAP-tag, biotin, Acrydite.

[0502] In a related embodiment, the fused droplets are recovered as described herein above.

[0503] In a related embodiment, the step of recovering the fused droplets as defined herein above further comprises the step of polymerizing the fused droplets.

[0504] For example, a hydrogel such as alginic acid can be polymerized by adding calcium ions. In other cases, a polymerization initiator, also known as a cross-linking agent, such as ammonium persulfate and TEMED for acrylamide, can be added to the droplets.

[0505] It will be understood by those skilled in the art that when the fused droplets are polymerized, the single-cell cDNA produced in step b) is contained in the polymerized droplets.

[0506] In some embodiments, polymerization is achieved by cross-linking.

[0507] In some embodiments, a cross-linking agent is added to the fused droplets, such as by diffusion.

[0508] In an alternative embodiment, the RT droplets comprise a cross-linking agent. It will be appreciated by those skilled in the art that therefore in the same embodiment, the fused droplets also comprise said cross-linking agent.

[0509] In some embodiments, the cross-linking agent used for polymerization is activated.

[0510] In particular, when the RT droplets contain a cross-linking agent, the cross-linking agent needs to be activated in order to polymerize.

[0511] In a certain embodiment, the crosslinking agent is activated by heat or UV.

[0512] In a related embodiment, for attaching the barcode to the cDNA in step c), the method of the present invention further comprises the step of dispersing the polymerized droplets containing the single cell cDNA into a plurality of reservoirs, wherein preferably one polymerized droplet is received in each reservoir. The reservoirs can be wells, typically a 96-well plate.

[0513] It is well known to the skilled person that small nucleic acids (eg barcode sequences) as well as enzymes (eg ligases) can be migrated into a polymer as defined herein above.

[0514] Thus, step c) of attaching a barcode sequence is applied to the cDNA contained in the polymer in the same manner as described herein above for attaching the cDNA on the particles. Thus, the steps of attaching a barcode on the cDNA are applied as those defined herein above in the embodiments relating to attaching the cDNA on the particles.

[0515] Thus, the barcode can be attached at either the 3' or 5' end.

[0516] In some embodiments, step c) of attaching a barcode sequence further comprises attaching a UMI sequence as defined herein above.

[0517] In some embodiments, step c) of attaching a barcode sequence is followed by a washing step to remove barcodes that are not attached to the cDNA.

[0518] In some embodiments, the method further comprises the step of removing unincorporated oligonucleotides. Removing unincorporated oligonucleotides is as defined herein above.

[0519] In some embodiments, the method further comprises the step of releasing the barcoded cDNA from the polymerized droplets. The cDNA can be released from the polymerized droplets by simple diffusion, photolysis or electrophoresis.

[0520] Preferably, the step of releasing the cDNA occurs after attaching the barcode sequence in step c).

[0521] In some embodiments, step c) of attaching the barcode sequence is followed by a step of recovering the barcoded cDNA. The recovered barcoded cDNA is then typically used for subsequent amplification and preparation of a sequencing library, as defined in the section "Amplification and Sequencing" herein above (see Fig.11 ).

[0522] Methods for genotyping single cells with phenotypes of interest

[0523] "Genotyping" generally refers to a method of determining the nucleic acid sequence of an individual using biochemical methods. In the context of the present invention, genotyping refers to determining the nucleic acid sequence of a single cell, particularly a single cell of interest.

[0524] "Phenotype" generally refers to a combination of observable characteristics or traits of an organism, such as its morphological, developmental, biochemical, or physiological properties. The phenotype results from the expression of the organism's genetic code (its genotype) and the influence of environmental factors and the interaction between the two. In the context of the present invention, the phenotype of a single cell refers to the presence or absence of a particular characteristic, such as the presence or absence of a particular antibody, or the presence or absence of a particular antigen, or the presence or absence of a particular activity, which can be observed or typically identified using a phenotypic assay.

[0525] An example of a phenotype of interest is the detection of a biological response. Among other things, the detection of an immune response can be monitored by detecting ADCC (antibody-dependent cell-mediated cytotoxicity) (3) induced by the secretion of antigen-specific antibodies (see Figure 1 ).

[0526] In biological events, immune responses can be monitored by measuring complement-dependent cytotoxicity (CDC) (6) induced by the secretion of antigen-specific antibodies (see Figure 2 ).

[0527] In biological events, the drug effect can be monitored by detecting ADC (antibody drug conjugate) induced by the secretion of antigen-specific antibodies (see Figure 3 ).

[0528] In biological events, including testing drug function, such as secretion of antigen-specific antibodies and induction of agonist / antagonist antibodies (see Figure 4 , 5 ).

[0529] In biological events, including testing drug function, such as competition assays (see Figure 6 ).

[0530] In biological events, including detection of drug function, such as detection of cell migration of reporter cells mediated by antibody secretion (see Figure 6 ). Such an assay can be used to track behavioral changes in reporter cells within the droplets. Target cells (1) expressing an antigen of interest and loaded with unreleased detectable markers will be co-encapsulated with IgG producing cells (2) and particles as single cell droplets. The droplets will be incubated under conditions that allow antibody secretion. The secreted specific antibodies will be relocated to the target cells. At the same time, the ligand / agonist diffuses from the particles with dynamics similar to IgG production and produces a ligand gradient. The reporter cells will migrate into the particles under the action of the ligand concentration (9). In the case of blocking antibodies (3), the progress of the reporter cells is changed. Then, migration can be used to evaluate the blocking / neutralizing activity of the antibodies.

[0531] Therefore, in the context of a method for genotyping single cells, single cells present in multiple reservoirs (e.g., typically in a 96-well plate reservoir, or in a microfluidic device reservoir) are first phenotyped and then genotyped. The inventors have developed a method in which, based on the use of a specific barcode in combination with a dye, it is possible to associate the genetic sequence of a barcoded single cell cDNA with the phenotype observed for the single cell after sequencing.

[0532] Thus, the present invention relates to a method for genotyping a single cell having a phenotype of interest, the method comprising:

[0533] - provide multiple reservoirs, and

[0534] a) for a plurality of reservoirs, providing at least one barcode sequence and at least one dye for each reservoir, wherein the at least one barcode sequence is associated with a color and a concentration of the at least one dye, and acquiring an image of the array to map the color and intensity of the at least one dye for each reservoir; and

[0535] b) for a plurality of reservoirs, providing at least one single cell for each reservoir and performing a phenotypic assay on the at least one single cell for each reservoir, and acquiring an image, thereby mapping the phenotype of the at least one single cell for each reservoir;

[0536] wherein step a) is performed before step b) or step b) is performed before step a); then

[0537] c) for a plurality of banks, comparing the phenotype of the single cell obtained in step b) with the color of the at least one dye obtained in step a) for each bank;

[0538] associating a color with said intensity; and

[0539] - for a plurality of reservoirs, reverse transcribing the nucleic acid of the at least one single cell for each reservoir to obtain single cell cDNA barcoded with the at least one barcode sequence, the at least one barcode sequence being associated with the color and concentration of the at least one dye; and

[0540] - correlating said genotype with said phenotype of said at least one single cell.

[0541] In a preferred embodiment, the method is a microfluidic method.

[0542] In some preferred embodiments, the plurality of reservoirs are contained in a microfluidic device comprising at least one microfluidic channel. Thus, in one embodiment, the method is a microfluidic method, the microfluidic method comprising the steps of providing a microfluidic device comprising a chip comprising at least one microfluidic channel and a plurality of reservoirs.

[0543] Therefore, the present invention also relates to a microfluidic method for genotyping a single cell having a phenotype of interest, the method comprising:

[0544] - providing a microfluidic device comprising a chip comprising a plurality of reservoirs, and

[0545] a) for a plurality of reservoirs, providing at least one barcode sequence and at least one dye for each reservoir, wherein the at least one barcode sequence is associated with a color and a concentration of the at least one dye, and acquiring an image of the array to map the color and intensity of the at least one dye for each reservoir; and

[0546] b) for a plurality of reservoirs, providing at least one single cell for each reservoir and performing a phenotypic assay on the at least one single cell for each reservoir, and acquiring an image, thereby mapping the phenotype of the at least one single cell for each reservoir;

[0547] wherein step a) is performed before step b) or step b) is performed before step a); then

[0548] c) for a plurality of banks, comparing the phenotype of the single cell obtained in step b) with the color of the at least one dye obtained in step a) for each bank;

[0549] associating a color with said intensity; and

[0550] - for a plurality of reservoirs, reverse transcribing the nucleic acid of the at least one single cell for each reservoir to obtain single cell cDNA barcoded with the at least one barcode sequence, the at least one barcode sequence being associated with the color and concentration of the at least one dye; and

[0551] - correlating said genotype with said phenotype of said at least one single cell.

[0552] In the context of microfluidic methods for genotyping single cells having a phenotype of interest, "microfluidic methods" and "microfluidic devices" are as defined in the section "Definitions" herein above.

[0553] A "barcode sequence" is as defined herein above in the section "Oligonucleotides".

[0554] "Multiple reservoirs" are as defined in the section "Microfluidic devices" herein above. In some embodiments, "at least one" in "at least one barcode sequence" and "at least one" in "at least one dye" refer to more than 1, more than 2, more than 3, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 barcode sequences and / or dyes, preferably 2, 3, 4 or 5 barcode sequences and / or dyes, more preferably 2, 3 or 4 barcode sequences and / or dyes, even more preferably 2 or 3 barcode sequences and / or dyes, for example 2 barcode sequences and / or dyes.

[0555] In some embodiments, the dye is a fluorescent dye. Fluorescent dyes are as defined herein above in the section "Single Cell".

[0556] In some preferred embodiments, at least one barcode and at least one dye are provided in one solution.

[0557] In some embodiments, at least one barcode and at least one dye are not linked to each other.

[0558] In some embodiments, the at least one barcode and the at least one dye are not covalently attached to each other.

[0559] In some embodiments, at least one barcode and at least one dye are not intentionally linked together using non-covalent linkage.

[0560] "Intentional" in this application refers to the fact that in some embodiments, at least one barcode and at least one dye are not exposed to specific conditions that induce non-covalent bonding, i.e., they are not modified using, for example, streptavidin and biotin to connect them to each other using non-covalent bonding. However, it will be understood by those skilled in the art that due to the properties of the at least one dye and the properties of the nucleic acid of the at least one barcode sequence, in some cases, the at least one dye and the at least one barcode sequence can be bound to each other by non-covalent interactions. These "non-covalent interactions" include electrostatic interactions, π-interactions, van der Waals forces, and hydrophobic interactions.

[0561] In some embodiments, at least one barcode is provided with at least one dye, wherein the at least one dye is provided at a known concentration.

[0562] In some embodiments, the concentration of the at least one dye is 0.1 nM to 100 μM, such as 1 nM to 50 μM, 1 nM to 30 μM, 1 μM to 50 μM, 1 μM to 30 μM, preferably 1 nM to 30 μM. It will be appreciated by those skilled in the art that the concentration used depends on the nature of the at least one dye.

[0563] In some preferred embodiments, when two barcode sequences are provided, two dyes are provided, such as when three barcode sequences are provided, three dyes are provided.

[0564] It will be understood by those skilled in the art that when at least one barcode sequence refers to a barcode sequence and at least one dye refers to a dye, the barcode sequence provided in the first reservoir is different from the barcode sequence provided in the other reservoir, and the dye provided with the barcode sequence in the first reservoir is different from the dye provided with the barcode sequence in the other reservoir and / or the concentrations of these dyes are different.

[0565] It will be understood by those skilled in the art that when at least one barcode sequence refers to two barcode sequences, and preferably at least one dye refers to two dyes, then at least one of the two barcode sequences provided in the first reservoir is different from at least one of the two barcode sequences provided in the other reservoir, and at least one of the two dyes provided with the two barcode sequences in the first reservoir is different from at least one of the two dyes provided with the two barcode sequences in the other reservoir and / or the concentration of at least one of the two dyes provided with the two barcode sequences in the first reservoir is different from the concentration of at least one of the two dyes provided with the two barcode sequences in the other reservoir.

[0566] The "acquiring an image" in step a) of the present application refers to acquiring an image by any method known to those skilled in the art, such as fluorescence microscopy, confocal microscopy or fluorescence imaging technology.

[0567] For further understanding, step a) is exemplified herein below, for two reservoirs (A and B), using two barcodes and two dyes in each reservoir, without limiting this example to step a). For example, barcode 1 is typically provided in solution with Alexa fluor 405 (purple), typically at a concentration of 10 μM. Barcode 2 is typically provided in solution with Alexa fluor 532 (green), typically at a concentration of 15 μM. Barcode 1 is combined with Alexa fluor 405 at a concentration of 10 μM and barcode 2 is combined with Alexa fluor 532 at a concentration of 15 μM. When these two barcodes, barcode 1 and barcode 2, are provided together in one reservoir (reservoir A), the colors and concentrations of the two dyes accompanying the barcodes are mixed together. When a fluorescence microscope image is typically acquired, the intensity and color of the fluorescence signal will be recorded for each reservoir (including reservoir A). Then, based on the reference ratio, it can be concluded that the color and intensity recorded for the mixture of these two dyes present in reservoir A is associated with the combination of barcodes 1 and 2.

[0568] When now in another reservoir, called reservoir B, barcode 1 is provided together with Alexa fluor 405 in a concentration of 10 μM and barcode 3 is provided together with Alexa 546 in a concentration of, for example, 20 μM, reservoir B will have another color and intensity than reservoir A. Therefore, based on the image acquired in step a), using the reference scale, it can be concluded that the color and intensity recorded for the dye mixture present in reservoir B are associated with the combination of barcodes 1 and 3.

[0569] Thus, the image of the array acquired in step a) enables to plot, for each of the plurality of reservoirs, the color and intensity of the at least one dye present in each reservoir. Furthermore, the color and intensity plotted for each reservoir is related to the at least one barcode sequence present in those reservoirs. Thus, for each of the plurality of reservoirs, a person skilled in the art is able to conclude from the image obtained in step a) that at least one barcode is provided in said reservoir.

[0570] The at least one barcode sequence and the at least one dye in step a) may be provided by any method known to those skilled in the art.

[0571] In some embodiments, for a plurality of reservoirs, at least one barcode sequence is attached to a surface of each reservoir in the plurality of reservoirs.

[0572] In a related embodiment, attaching the at least one barcode sequence to the surface of the reservoir does not affect the at least one dye in the solution. The phrase "does not affect the at least one dye in the solution" means that the dye remains in the solution.

[0573] Methods for attaching nucleic acids, such as at least one barcode sequence, to a surface are well known to those skilled in the art and are described, for example, in US 5412087. Those methods are further described in the section "Oligonucleotides" herein above, in the context of attaching at least one oligonucleotide to at least one first type of particle.

[0574] Thus, in some embodiments, for a plurality of reservoirs, at least one barcode sequence is covalently or non-covalently linked to the surface of the reservoir. "Non-covalently linked", "covalently linked", "streptavidin" are as defined in the section "Oligonucleotides" herein above.

[0575] In a preferred embodiment, the non-covalent bond is a streptavidin-biotin linkage.

[0576] Non-covalent bonds are well known to those skilled in the art, such as streptavidin-biotin bonds. Thus in one example, the surface of the reservoir is coated with, for example, a hydrogel, as defined in the section "Oligonucleotides" herein above, the hydrogel may be functionalized with streptavidin, and at least one barcode sequence may be functionalized with biotin.

[0577] In some embodiments, where at least one barcode sequence refers to more than one barcode sequence, one skilled in the art will appreciate that in some embodiments, the barcode sequences are sequentially attached to the surface of the reservoir. Thus, the first barcode sequence is attached to the surface of the reservoir as described herein above, then the second barcode sequence is attached to the first barcode sequence, and finally the third barcode sequence is attached to the second barcode sequence, and so on, depending on the number of barcode sequences.

[0578] In an alternative embodiment, prior to attaching the (at least one) barcode sequence to the surface of the reservoir, the barcode sequences are attached to each other resulting in one barcode sequence.

[0579] In other embodiments, at least one barcode sequence is further attached to a UMI sequence, wherein the UMI sequence is as defined in the section “Oligonucleotides” described herein above.

[0580] Methods for attaching nucleic acids to each other, or attaching two barcode sequences or a UMI sequence to a barcode sequence are well known to those of skill in the art and include, but are not limited to, using a ligase and / or using annealing or primer extension methods.

[0581] Thus, in some embodiments, the solution in which at least one barcode sequence and at least one dye are provided further comprises a ligase. Examples of ligases are given in the section "Oligonucleotides" herein above.

[0582] Independent of the type of bond used to attach the at least one barcode to the surface of the reservoir, the barcode sequence may also comprise at least one linker sequence.

[0583] In some embodiments, the barcode sequence is preferably attached to the surface using either the 5' end or the 3' end, preferably the 5' end.

[0584] It will be understood by those skilled in the art that in some embodiments, wherein at least one barcode sequence refers to more than one barcode sequence, the first barcode sequence attached to the surface of the well (i.e., the barcode sequence located at the 5' end or the 3' end, preferably the 5' end) comprises at least one adapter sequence at its 5' end or the 3' end (preferably the 5' end).

[0585] In some embodiments, a "linker sequence" is a sequence that is optionally linked to a reservoir surface by means of its at least one barcode sequence.

[0586] "Optionally linked" in the present application refers to the possibility that at least one barcode sequence can be released so that at least one barcode sequence is preferably attached to at least one primer sequence, as further described below in the present application.

[0587] Preferably, the linker sequence is a cleavable linker sequence as defined herein above in the section "Oligonucleotides".

[0588] Therefore, in some embodiments, the methods of the present invention further comprise the step of releasing at least one barcode sequence bound to the surface of the reservoir.

[0589] In some embodiments, as further described herein below, when the method is performed in a droplet, at least one barcode sequence is released, in particular into at least one RT droplet.

[0590] In some embodiments, at least one barcode sequence is released into at least one RT droplet by contacting the RT droplet with a surface of a reservoir.

[0591] In some embodiments, the RT droplet is contacted with the surface by wetting.

[0592] Thus, in some embodiments, at least one barcode sequence is released into at least one RT droplet by wetting.

[0593] In this application "wetting" refers to changing the surface energy of a semi-solid phase or a liquid phase on a solid phase.

[0594] Furthermore, in some embodiments, prior to reverse transcribing the nucleic acid in the single cell, at least one barcode sequence attached to the surface of the reservoir is released from the surface.

[0595] Furthermore, in some embodiments, prior to lysing the at least one single cell, at least one barcode sequence attached to the surface of the reservoir is released from the surface.

[0596] When performed in droplets, in some embodiments, at least one barcode sequence attached to the surface of the reservoir is released from the surface prior to fusion of at least one single-cell droplet with the RT droplet.

[0597] In some embodiments, at least one barcode can be released using any method, such as enzymes, nucleophilic / basic reagents, reducing agents, photoirradiation, electrophilic / acidic reagents, organometallic and metallic reagents, and oxidizing agents.

[0598] In some embodiments, at least one barcode can be released using enzymatic and / or photocleavage. For example, an endonuclease can be used to cleave a linker sequence or any other sequence to release at least one barcode sequence from the surface of the reservoir.

[0599] In other embodiments, releasing at least one barcode sequence refers to breaking a bond, such as a streptavidin-biotin bond, as defined in the section "Oligonucleotides" herein above.

[0600] In some embodiments, at least one barcode is released by enzymatic digestion of streptavidin.

[0601] It will be appreciated by those skilled in the art that the components required to release the barcode sequence can be provided by any method known to those skilled in the art, can be in each of a plurality of reservoirs, or when performed in droplets, for example can be in RT droplets, such that at least one barcode sequence is typically released when the RT droplets are contacted with a surface.

[0602] In step b) of the method, for a plurality of banks, at least one single cell is provided in each bank.

[0603] "At least one single cell" is defined herein in the above section "single cell", and preferably refers to one single cell.

[0604] In the present context "phenotypic assay" refers to a method that allows the identification of the phenotype of a cell, more specifically the phenotype of a single cell."Phenotype" is as defined herein above.

[0605] In some embodiments, the phenotypic assay is selected from detecting a DNA sequence; detecting an RNA sequence; detecting a molecule selected from the group consisting of a protein, a carbohydrate, a lipid, and any combination thereof; detecting a small molecule; detecting an activity; detecting an ion concentration; detecting an ion potential; and detecting an oxidation-reduction potential.

[0606] According to the phenotypic assay, at least one single cell can be assayed for the selected parameter using any suitable assay method, which can be qualitative and / or quantitative. Suitable detection methods may include spectroscopic methods, electrical methods, fluid dynamic methods, imaging methods and / or biological methods, etc., particularly those suitable or applicable to the analysis of single cells. These methods may involve detecting single or multiple values, values ​​that are time-dependent or time-independent (e.g., steady state or endpoint) and / or average values ​​or (time and / or space) distribution values, etc. These methods can measure and / or output analog and / or digital values.

[0607] "Spectroscopic methods" may generally include detecting any property of light (or wave-like particles), particularly properties that are altered by interaction with a sample. Suitable spectroscopic methods may include absorption, luminescence (including photoluminescence, chemiluminescence and electrochemiluminescence), magnetic resonance (including nuclear and electron spin resonance), scattering (including light scattering, electron scattering and neutron scattering), diffraction, circular dichroism and optical rotation, among others.

[0608] Suitable "photoluminescence methods" may include bioluminescence resonance energy transfer (BRET), fluorescence intensity (FLINT), fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), fluorescence lifetime (FLT), total internal reflection fluorescence (TIRF), fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence activated cell sorting (FACS), and phosphorescence and other analogs thereof, among others.

[0609] As will be appreciated by those skilled in the art, in some embodiments, performing a phenotypic assay may also require exposing the cells to an agent.

[0610] Thus, in some embodiments, the single cell is exposed to one or more agents before, during, or after the parameter is measured.

[0611] In some embodiments, the single cell is exposed to one or more agents prior to providing it in step b).

[0612] In some embodiments, reagents include, but are not limited to, chemical / biological cell conditioning agents, detection / assay reagents, solvents, buffers, culture media, washing solutions.

[0613] "Chemical modulators or biological modulators" may include any agent for detecting an interaction with a cell. The interaction typically includes specific binding to the particle and / or any detectable genotypic or phenotypic effect on the particle (or modulator). Chemical / biological modulators may include ligands (e.g., antagonists, agonists, hormones, etc.), ions, gases, carbohydrates, lipids, organics, polymers, proteins, small molecules, or nucleic acids (particularly siRNA) that interact with a receptor.

[0614] "Detection / assay reagents" may include stains, dyes, labels, enzymes, substrates, cofactors and / or specific binding partners (SBPs), and the like.

[0615] In some embodiments, acquiring an image in step b) refers to acquiring an image using any method known to those skilled in the art that is suitable for the phenotypic assay used, typically a spectroscopic method. In particular, acquiring an image refers to acquiring an image using a bright field microscope or a fluorescence microscope with an array or linear camera.

[0616] Those skilled in the art will know how to adapt the phenotypic assays performed using the reagents used and the techniques for acquiring images.

[0617] The images acquired in step b) map for each reservoir the phenotype of the single cells contained in each reservoir.

[0618] Thus, in step c), based on the image obtained in step a) and the image obtained in step b), for a plurality of reservoirs, a person skilled in the art is able to associate the phenotype obtained in step b) in one reservoir with the color and intensity of at least one dye obtained in step a) for the same reservoir, wherein the color and intensity of at least one dye obtained in step a) are associated with at least one barcode sequence provided in said same reservoir.

[0619] Thus, for a plurality of banks, the skilled person is thus able in step c) to associate the phenotype of at least one single cell observed in one bank with at least one barcode sequence provided in said same bank.

[0620] In some embodiments, the method further comprises at least 1, 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, at least 11 steps selected from the following steps, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 steps:

[0621] - at least one cleanup step,

[0622] - for a plurality of reservoirs, providing at least one oligonucleotide for each reservoir, wherein the at least one oligonucleotide comprises a primer sequence,

[0623] - for a plurality of reservoirs, attaching the at least one barcode sequence to the at least one oligonucleotide for each reservoir,

[0624] - for a plurality of reservoirs, providing a lysis composition for each reservoir,

[0625] - for a plurality of banks, lysing the at least one single cell for each bank,

[0626] - for a plurality of reservoirs, providing a reverse transcriptase and a reverse transcriptase composition for each reservoir,

[0627] - for a plurality of reservoirs, hybridizing at least some of the single-cell nucleic acids for each reservoir with the at least one barcoded oligonucleotide,

[0628] - recovering the barcoded single cell cDNA,

[0629] - removing unincorporated oligonucleotides from said barcoded cDNA,

[0630] - amplifying the barcoded single cell cDNA,

[0631] - Sequencing the barcoded single cell cDNA.

[0632] These additional steps are described in the context of "microfluidic methods for barcoding single-cell nucleic acids" herein above. Therefore, those skilled in the art will understand that the embodiments and features throughout the specification related to the above steps can be fully applied to these steps.

[0633] For example, descriptions referring to “at least one oligonucleotide” in the sections “Preparation of droplets,” “Oligonucleotides,” “Reverse transcription,” and “Microfluidic methods for barcoding single-cell nucleic acids” are fully applicable to the section entitled “Methods for genotyping single cells having a phenotype of interest.” Descriptions referring to “primers” or “primer sequences” in the sections “Definitions,” “Reverse transcription,” “Oligonucleotides,” and “Microfluidic methods for barcoding single-cell nucleic acids” are fully applicable to the section entitled “Methods for genotyping single cells having a phenotype of interest.”

[0634] It will be understood by those skilled in the art that the method for "attaching at least one barcode sequence to at least one oligonucleotide in each reservoir" is equivalent to the method for "attaching a barcoded sequence to a single cell cDNA" as described in the context of step c) of the method for barcoding a single cell nucleic acid or the method for "attaching at least one barcode sequence to a UMI sequence" or "attaching barcode sequences to each other" as described above herein. Thus, methods for attaching at least one barcode sequence to at least one oligonucleotide are well known to those skilled in the art and typically include, but are not limited to, the use of a ligase and / or the use of an annealing or primer extension method. Examples of ligases are given in the section "Oligonucleotides" above herein. In some embodiments, the barcode sequence is preferably attached to the at least one oligonucleotide at the 5' end of the at least one oligonucleotide resulting in the oligonucleotide comprising the barcode sequence and the primer sequence from 5' to 3'. Thus, if the at least one oligonucleotide is connected to the at least one particle, the at least one oligonucleotide is preferably released before attaching the at least one barcode sequence.

[0635] In addition, the descriptions in the sections entitled “Definitions,” “Cell lysis,” “Reverse transcription,” “Oligonucleotides,” and “Microfluidic methods for barcoding single-cell nucleic acids” that relate to “lysis compositions” are fully applicable to the section entitled “Methods for genotyping single cells having a phenotype of interest.” Similarly, the steps in the sections entitled “Cell lysis,” “Reverse transcription,” “Oligonucleotides,” and “Microfluidic methods for barcoding single-cell nucleic acids” that relate to “lysing at least one single cell” are fully applicable to the section entitled “Methods for genotyping single cells having a phenotype of interest.”

[0636] Descriptions concerning "reverse transcriptase" and "reverse transcriptase composition" or "reverse transcription step" in the sections "Definitions", "Preparation of RT droplets", "Reverse transcription", "Oligonucleotides", and "Microfluidic methods for barcoding single-cell nucleic acids" are fully applicable to the section entitled "Methods for genotyping single cells with a phenotype of interest".

[0637] The descriptions concerning the "hybridization step" in the sections "Definitions" and "Microfluidic methods for barcoding single-cell nucleic acids" are fully applicable to the section entitled "Methods for genotyping single cells with a phenotype of interest."

[0638] In some embodiments, recovering barcoded single-cell cDNA in the present application refers to separating the barcoded cDNA generated by reverse transcription from a reservoir.

[0639] The description of the steps involving "Removal of Unincorporated Oligonucleotides" in the section "Microfluidic Methods for Barcoding Single-Cell Nucleic Acids" is fully applicable to the section entitled "Methods for Genotyping Single Cells with a Phenotype of Interest".

[0640] The descriptions of the steps involving "Amplifying barcoded single-cell cDNA" and "Sequencing barcoded single-cell cDNA" in the "Amplification and Sequencing" section are fully applicable to the section entitled "Methods for genotyping single cells with a phenotype of interest."

[0641] It will be appreciated by those skilled in the art that the single cells in step b) contain nucleic acid.

[0642] As described herein above, it will be understood by those skilled in the art that the step of lysing at least one single cell in a reservoir results in nucleic acid from the single cell entering the reservoir.

[0643] In some embodiments, the step of lysing the cells occurs before the step of transcribing. In some preferred embodiments, the step of lysing the cells occurs after step b) of performing a phenotypic assay.

[0644] According to the above, in some embodiments, a microfluidic method for genotyping a single cell having a phenotype of interest comprises:

[0645] - providing a microfluidic device comprising a chip, the chip comprising a plurality of reservoirs, and

[0646] 1. for a plurality of reservoirs, providing at least one barcode sequence and at least one dye for each reservoir, wherein the at least one barcode sequence is associated with a color and a concentration of the at least one dye, and acquiring an image of the array to map the color and intensity of the at least one dye for each reservoir; and

[0647] 2. For a plurality of reservoirs, providing at least one single cell for each reservoir and performing a phenotypic assay on the at least one single cell for each reservoir, and acquiring an image,

[0648] thereby mapping said phenotype of said at least one single cell for each reservoir;

[0649] wherein step a) is performed before step b) or step b) is performed before step a); then

[0650] 3. For a plurality of banks, comparing the phenotype of the single cell obtained in step b) with the color of the at least one dye obtained in step a) for each bank.

[0651] associating a color with said intensity; and

[0652] -optionally, for a plurality of reservoirs, providing at least one oligonucleotide for each reservoir, wherein the at least one oligonucleotide comprises a primer sequence,

[0653] -optionally, for a plurality of reservoirs, attaching said at least one barcode sequence to said at least one oligonucleotide for each reservoir,

[0654] - optionally, for a plurality of reservoirs, providing a lysis composition for each reservoir,

[0655] - optionally, for a plurality of banks, lysing the at least one single cell for each bank,

[0656] -optionally, for a plurality of reservoirs, providing a reverse transcriptase and a reverse transcriptase composition for each reservoir,

[0657] - optionally, for a plurality of depots, hybridizing at least some of said single cell nucleic acids to said at least one barcoded oligonucleotide for each depot,

[0658] - Optionally, for a plurality of reservoirs, reverse transcribing nucleic acids of each reservoir of said at least one single cell to obtain single cell cDNA barcoded with said at least one barcode sequence associated with the color and concentration of at least one dye;

[0659] -optionally, recovering the barcoded single cell cDNA,

[0660] - optionally, removing unincorporated oligonucleotides from said barcoded cDNA,

[0661] -optionally, amplifying said barcoded single cell cDNA,

[0662] - optionally, sequencing the barcoded single cell cDNA, and

[0663] - associating the genotype with the phenotype of the at least one single cell.

[0664] In some embodiments, a method for genotyping a single cell having a phenotype of interest is performed in a droplet. A droplet is as defined in the section "Droplet" herein above.

[0665] Therefore, at least one single cell provided in a droplet is referred to as a single-cell droplet. The descriptions of "single-cell droplets" in previous sections such as "droplets", "single cells", and "microfluidic methods for barcoding single-cell nucleic acids" are fully applicable to the section called "methods for genotyping single cells with a phenotype of interest".

[0666] In a related embodiment, at least one oligonucleotide and a reverse transcriptase are provided in a droplet, which is referred to herein as an RT droplet. Therefore, the descriptions of "RT droplets" in previous sections such as "Definitions", "Droplets", "Preparation of droplets", "Single cells", "Cell lysis", "Reverse transcription", "Oligonucleotides", and "Microfluidic methods for barcoding single cell nucleic acids" are fully applicable to the section entitled "Methods for genotyping single cells having a phenotype of interest".

[0667] As will be appreciated by those skilled in the art, in certain embodiments, RT droplets or single-cell droplets may be provided first, as described herein above in the context of "Microfluidic methods for barcoding single-cell nucleic acids". Thus, in some embodiments, the method comprises the steps of:

[0668] - injecting a carrier fluid into an inlet of the microfluidic channel, the carrier fluid comprising a plurality of first type droplets dispersed in the carrier fluid, wherein the first type droplets are 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 a single cell, wherein the single cell comprises a single-cell nucleic acid,

[0669] - for a plurality of reservoirs, a first migration step, wherein at least one droplet of the first type in the plurality of droplets is caused to enter a reservoir of the plurality of reservoirs,

[0670] - injecting a carrier fluid into an inlet of the microfluidic channel, the carrier fluid comprising a plurality of droplets of a second type dispersed in the carrier fluid, wherein the droplets of the second type are 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,

[0671] - for a plurality of reservoirs, a second migration step, wherein at least a portion of at least one droplet of the second type enters a reservoir of the plurality of reservoirs.

[0672] The description of these steps in the section titled "Microfluidic Methods for Barcoding Single-Cell Nucleic Acids" is fully applicable to the section titled "Microfluidic Methods for Genotyping Single Cells with Phenotypes of Interest."

[0673] In some embodiments, those steps may be performed before step b) of performing a phenotypic assay on each reservoir on the at least one single cell. It will be appreciated by those skilled in the art that the phenotypic assay of step b) as described herein above may be performed on a single cell when present in a droplet.

[0674] As will be further appreciated by one skilled in the art, phenotypic assays may be performed when single-cell droplets are provided in multiple reservoirs, or when both single-cell droplets and RT droplets are provided in multiple reservoirs.

[0675] In some embodiments, in the first migration step, at least one droplet of the first type in the plurality of droplets enters one of the plurality of reservoirs by buoyancy, hydrodynamic force or by a physical field, preferably buoyancy.

[0676] As will be further understood by those skilled in the art, when the method is performed in a droplet, the method further comprises the following steps:

[0677] - For a plurality of reservoirs, the at least one droplet of the first type merges with the at least one droplet of the second type in each reservoir or at its edge, resulting in a fused droplet.

[0678] In a preferred embodiment, the fusion step is preferably performed after step b) of performing a phenotypic determination.

[0679] The description of the fusion step in previous sections such as "Microfluidic methods for barcoding single-cell nucleic acids" is fully applicable to the section entitled "Microfluidic methods for genotyping single cells with phenotypes of interest".

[0680] In some embodiments, when the method is performed in droplets, at least one barcode sequence and at least one dye are provided in each reservoir as defined herein above. Thus, in some embodiments, after acquiring an image of the at least one dye in step a), the at least one barcode sequence is incorporated into the RT droplets by, for example, wetting as described herein above.

[0681] Alternatively, in some embodiments, at least one barcode sequence and at least one dye are provided with a reverse transcriptase and at least one oligonucleotide in an RT droplet of a plurality of RT droplets.

[0682] In related embodiments, at least one barcode sequence is attached to at least one oligonucleotide in the RT droplet, resulting in at least one barcoded oligonucleotide. The method of attaching a barcode sequence to an oligonucleotide is as described herein above. In some related embodiments, the RT droplet further comprises a ligase.

[0683] When the “method for genotyping a single cell having a phenotype of interest” is performed in a droplet, the method further comprises the following steps after step c):

[0684] - for each fused droplet, hybridizing at least some single-cell nucleic acids from at least one cell with at least one barcoded oligonucleotide,

[0685] - In each fused droplet, at least some of the single-cell nucleic acids present in the fused droplet are reverse transcribed to generate barcoded single-cell cDNA, wherein the barcode sequence encodes the identity of the single cell.

[0686] The descriptions involving hybridization and reverse transcription steps in previous sections such as "Microfluidic methods for barcoding single-cell nucleic acids" are fully applicable to the section entitled "Microfluidic methods for genotyping single cells with phenotypes of interest."

[0687] When performed in droplets, the method of the present invention further comprises a step of recovering fused droplets at the outlet of the channel after the reverse transcription step. In the present application, "recovering fused droplets" refers to collecting microfluidic droplets containing barcoded cDNA, as described in the section "Microfluidic methods for barcoding single-cell nucleic acids" above.

[0688] With respect to "at least one oligonucleotide", in some embodiments, the at least one oligonucleotide is linked to at least one first type of particle. In some embodiments, the at least one oligonucleotide is introduced into the reservoir or droplet, in particular into the RT droplet, by initially linking the at least one oligonucleotide to at least one first type of particle.

[0689] Therefore, the descriptions in previous sections such as "Oligonucleotides" and "Microfluidic methods for barcoding single-cell nucleic acids" involving at least one oligonucleotide attached to at least one first type of particle are fully applicable to the section entitled "Microfluidic methods for genotyping single cells with a phenotype of interest."

[0690] Thus, in some embodiments, at least one oligonucleotide is covalently or non-covalently linked to the at least one first type of particle, wherein "covalently linked" or "non-covalently linked" and "at least one first type of particle" are as defined in the section "Oligonucleotide" herein above.

[0691] Independently of the type of bond used to connect the at least one type of oligonucleotide to the first type of particle, the at least one type of oligonucleotide may further comprise at least one linker sequence, wherein the linker sequence is as defined in the section "Oligonucleotides" herein above.

[0692] Therefore, in other embodiments, "at least one type of oligonucleotide" or simply "oligonucleotide" further comprises at least one linker sequence, and the linker sequence is preferably included at the 5' end or the 3' end, more preferably at the 5' end.

[0693] Those skilled in the art will appreciate that in some embodiments, at least one oligonucleotide is bound to the first type of particle via the 3' end or the 5' end (preferably the 5' end).

[0694] In some embodiments, the method of the invention further comprises the step of releasing the at least one oligonucleotide bound to the at least one first type of particle from said particle after its incorporation into the reservoir or RT droplet.

[0695] Furthermore, in some embodiments, prior to reverse transcribing the single-cell nucleic acid, at least one oligonucleotide initially bound to at least one first type of particle is released from the at least one first type of particle.

[0696] Furthermore, in some embodiments, at least one oligonucleotide initially bound to at least one first type of particle is released from the at least one first type of particle either before or after attachment of at least one barcode.

[0697] The step of releasing at least one oligonucleotide may occur before or after the fusion of the single-cell droplet with the RT droplet; preferably before the fusion of the droplets.

[0698] The step of releasing the at least one oligonucleotide may occur after lysing the single cell but before reverse transcribing the single cell nucleic acid.

[0699] It will be understood by those skilled in the art that the number of oligonucleotides present in a reservoir or droplet will be adapted to the number of different nucleic acids, in particular the number of RNAs, which are transcribed and barcoded in a single cell. The number of oligonucleotides in a reservoir or droplet will be adjusted in the same manner as the adjustment of oligonucleotides in RT droplets described in the section "Oligonucleotides". Thus, the "at least one" in the phrase "at least one oligonucleotide" refers to the number of different oligonucleotides present in a reservoir or droplet, wherein one of the at least one oligonucleotides has a primer sequence different from another oligonucleotide. The "at least one" in the phrase "at least one oligonucleotide" is as defined in the section "Oligonucleotides".

[0700] The concentration of the at least one oligonucleotide is as defined herein above in the section "Oligonucleotides".

[0701] The concentration of the at least one oligonucleotide is at least 10 nM, preferably at least 100 nM.

[0702] Throughout this application, the term "and / or" is a grammatical conjunction that is interpreted as a possibility that one or more situations involving what it is connected may occur. For example, the wording "error detection and / or correction" in the phrase "error detection and / or correction may be performed on the sequence" means that error detection may be performed on the sequence and error correction may be performed on the sequence or error detection may be performed on the sequence or error correction may be performed on the sequence.

[0703] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references, such as multiple referents, unless the context clearly dictates otherwise.

[0704] Throughout this application, the term "comprising" should be interpreted as including all specifically mentioned features as well as optional, additional and unspecified features. As used in this application, the use of the term "comprising" also discloses embodiments in which there are no other features than the specifically mentioned features (i.e., "consisting of ...").

[0705] Throughout the specification, features described in one section are fully applicable to other sections of the specification. Therefore, the present invention also includes the combination of embodiments described in different sections of the specification.

[0706] This application also includes the following items:

[0707] 1. A method for genotyping a single cell having a phenotype of interest, the method comprising:

[0708] - provide multiple reservoirs, and

[0709] a) for a plurality of reservoirs, providing at least one barcode sequence and at least one dye for each reservoir, wherein the at least one barcode sequence is associated with a color and a concentration of the at least one dye, and acquiring an image of the array to map the color and intensity of the at least one dye for each reservoir; and

[0710] b) for a plurality of reservoirs, providing at least one single cell for each reservoir and performing a phenotypic assay on the at least one single cell for each reservoir, and acquiring an image, thereby mapping the phenotype of the at least one single cell for each reservoir;

[0711] wherein step a) is performed before step b) or step b) is performed before step a); then

[0712] c) for a plurality of reservoirs, correlating for each reservoir the phenotype of said single cell obtained in step b) with said color and said intensity of said at least one dye obtained in step a); and

[0713] - for a plurality of reservoirs, reverse transcribing the nucleic acid of the at least one single cell for each reservoir to obtain single cell cDNA barcoded with the at least one barcode sequence, the at least one barcode sequence being associated with the color and concentration of the at least one dye; and

[0714] - correlating said genotype with said phenotype of said at least one single cell.

[0715] 2. The microfluidic method according to item 1, wherein the method further comprises at least one step selected from the following:

[0716] - at least one cleanup step,

[0717] - for a plurality of reservoirs, providing at least one oligonucleotide for each reservoir, wherein the at least one oligonucleotide comprises a primer sequence,

[0718] - for a plurality of reservoirs, attaching the at least one barcode sequence to the at least one oligonucleotide for each reservoir,

[0719] - for a plurality of reservoirs, providing a lysis composition for each reservoir,

[0720] - for a plurality of banks, lysing the at least one single cell for each bank,

[0721] - for a plurality of reservoirs, providing a reverse transcriptase and a reverse transcriptase composition for each reservoir,

[0722] - for a plurality of reservoirs, hybridizing at least some of the single-cell nucleic acids with at least one barcoded oligonucleotide for each reservoir,

[0723] - recovering the barcoded single cell cDNA,

[0724] - removing unincorporated oligonucleotides from said barcoded cDNA,

[0725] - amplifying the barcoded single cell cDNA,

[0726] - Sequencing the barcoded single cell cDNA.

[0727] 3. A microfluidic method according to item 1 or 2, wherein the method is a microfluidic method comprising the step of providing a microfluidic device, the microfluidic device comprising a chip, the chip comprising at least one microfluidic channel and a plurality of reservoirs.

[0728] 4. The microfluidic method according to item 3, wherein, for each reservoir, the at least one single cell is provided in a single-cell droplet.

[0729] 5. The microfluidic method according to item 3 or 4, wherein the reverse transcriptase, the reverse transcriptase composition and the at least one oligonucleotide are provided in RT droplets for each reservoir.

[0730] 6. The microfluidic method according to any one of items 1 to 5, wherein, for a plurality of reservoirs, the at least one barcode sequence is attached to the surface of each reservoir of the plurality of reservoirs.

[0731] 7. The microfluidic method according to item 6, wherein the at least one barcode sequence is released before transcription.

[0732] 8. A microfluidic method for barcoding single-cell nucleic acids, the method comprising:

[0733] - providing a microfluidic device comprising a chip, the chip comprising at least one microfluidic channel and a plurality of reservoirs,

[0734] - injecting a carrier fluid into an inlet of the microfluidic channel, the carrier fluid comprising a plurality of first type droplets dispersed in the carrier fluid, wherein the first type droplets are 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 a single cell, wherein the single cell comprises a single-cell nucleic acid,

[0735] - for a plurality of reservoirs, a first migration step, wherein at least one droplet of the first type in the plurality of droplets is moved into a reservoir of the plurality of reservoirs by buoyancy,

[0736] - injecting a carrier fluid into an inlet of the microfluidic channel, the carrier fluid comprising a plurality of droplets of a second type dispersed in the carrier fluid, wherein the droplets of the second type are 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,

[0737] - for a plurality of reservoirs, a second migration step, wherein at least a portion of at least one droplet of the second type enters a reservoir of the plurality of reservoirs,

[0738] - for a plurality of reservoirs, said at least one droplet of said first type merges with said at least one droplet of said second type in each reservoir or at its edge, thereby producing a fused droplet,

[0739] - and further comprising the following steps:

[0740] a) for each fused droplet, hybridizing at least some of the single-cell nucleic acid to the at least one oligonucleotide in the fused droplet,

[0741] b) in each fused droplet, reverse transcribing at least some of the single-cell nucleic acid present in the fused droplet, thereby producing single-cell cDNA, and

[0742] c) attaching at least one barcode sequence to the single cell cDNA obtained in step b), wherein the at least one barcode sequence encodes the identity of the single cell,

[0743] or

[0744] - It also includes the following steps:

[0745] a) for each fused droplet, hybridizing at least some of the single-cell nucleic acid from at least one cell to the at least one oligonucleotide in the fused droplet, wherein the at least one oligonucleotide comprises at least one barcode sequence,

[0746] b) in each fused droplet, reverse transcribing at least some of the single-cell nucleic acids present in the fused droplet, thereby generating barcoded single-cell cDNA, wherein the at least one barcode sequence encodes the identity of the single cell.

[0747] 9. The microfluidic method according to item 8, wherein the RT droplet further comprises at least one particle of the first type to which the at least one oligonucleotide is bound.

[0748] 10. The microfluidic method according to item 8 or 9, wherein the RT droplets further comprise a polymer.

[0749] 11. A microfluidic method according to any one of items 8 to 10, wherein the plurality of droplets of the first type have an average volume of 1 pL to 3000 pL.

[0750] 12. A microfluidic method according to any one of items 8 to 11, wherein the plurality of droplets of the second type have an average volume of 10 pL to 5000 pL.

[0751] 13. A microfluidic method according to any one of items 8 to 12, wherein the first migration step of at least one droplet of the first type results in an occupation of 90% to 100% of the total number of droplets in the reservoir and / or results in a capture efficiency of 80% to 100% of the injected droplets of the first type.

[0752] 14. A microfluidic method according to any one of items 8 to 13, wherein the at least a portion of the at least one droplet of the second type refers to 90% to 100% of the total volume of the at least one droplet of the second type or refers to 10% to 30% of the total volume of the at least one droplet of the second type.

[0753] 15. A microfluidic method according to any one of items 8 to 14, wherein the fusion step results in a fusion efficiency between the first type of droplets and the second type of droplets of 90% to 100%.

Claims

1. A microfluidic method for barcoding single-cell nucleic acids, the method comprising: - providing a microfluidic device, the microfluidic device comprising a chip; - Load the following into the chip: at least one first type of droplet dispersed in a carrier fluid, wherein the at least one first type of droplet comprises a cell comprising a single-cell nucleic acid, and at least one droplet of a second type dispersed in a carrier fluid, wherein the at least one droplet of the second type is a reverse transcription (RT) droplet comprising a reverse transcriptase; - fusing at least one droplet of the first type with at least one droplet of the second type in the chip, thereby producing a fused droplet; and - It also includes the following steps: a) for each fused droplet, hybridizing at least some of the single-cell nucleic acid to the at least one oligonucleotide in the fused droplet, b) in each fused droplet, reverse transcribing at least some of the single-cell nucleic acid present in the fused droplet to produce single-cell cDNA, and c) attaching at least one barcode sequence to the single cell cDNA obtained in step b), wherein the at least one barcode sequence encodes the identity of the single cell, or - It also includes the following steps: a) for each fused droplet, hybridizing at least some of the single-cell nucleic acids from at least one cell to the at least one oligonucleotide in the fused droplet, wherein The at least one oligonucleotide comprises at least one barcode sequence, and b) in each fused droplet, reverse transcribing at least some of the single-cell nucleic acids present in the fused droplet, thereby generating barcoded single-cell cDNA, wherein the at least one barcode sequence encodes the identity of the single cell.

2. A method for genotyping a single cell having a phenotype of interest, the method comprising: - provide multiple reservoirs, and a) for a plurality of reservoirs, providing at least one barcode sequence and at least one dye for each reservoir, wherein the at least one barcode sequence is associated with a color and a concentration of the at least one dye, and acquiring an image of the array to map the color and intensity of the at least one dye for each reservoir; and b) for a plurality of reservoirs, providing at least one single cell for each reservoir and performing a phenotypic assay on the at least one single cell for each reservoir, and acquiring an image, thereby mapping the phenotype of the at least one single cell for each reservoir; wherein step a) is performed before step b) or step b) is performed before step a); then c) for a plurality of reservoirs, correlating for each reservoir the phenotype of said single cell obtained in step b) with said color and said intensity of said at least one dye obtained in step a); and - for a plurality of reservoirs, reverse transcribing the nucleic acid of the at least one single cell for each reservoir to obtain single cell cDNA barcoded with the at least one barcode sequence, the at least one barcode sequence being associated with the color and concentration of the at least one dye; and - correlating said genotype with said phenotype of said at least one single cell.

3. The microfluidic method according to claim 2, wherein the method further comprises at least one step selected from the following: - at least one cleanup step, - for a plurality of reservoirs, providing at least one oligonucleotide for each reservoir, wherein the at least one oligonucleotide comprises a primer sequence, - for a plurality of reservoirs, attaching the at least one barcode sequence to the at least one oligonucleotide for each reservoir, - for a plurality of reservoirs, providing a lysis composition for each reservoir, - for a plurality of banks, lysing the at least one single cell for each bank, - for a plurality of reservoirs, providing a reverse transcriptase and a reverse transcriptase composition for each reservoir, - for a plurality of reservoirs, hybridizing at least some of the single-cell nucleic acids with at least one barcoded oligonucleotide for each reservoir, - recovering the barcoded single cell cDNA, - removing unincorporated oligonucleotides from said barcoded cDNA, - amplifying the barcoded single cell cDNA, - Sequencing the barcoded single cell cDNA.

4. The microfluidic method according to claim 2 or 3, wherein the method is a microfluidic method comprising the step of providing a microfluidic device, the microfluidic device comprising a chip, the chip comprising at least one microfluidic channel and a plurality of reservoirs.

5. The microfluidic method according to claim 4, wherein: For each reservoir, the at least one single cell is provided in a single-cell droplet.

6. The microfluidic method according to claim 4 or 5, wherein: For each reservoir, the reverse transcriptase, the reverse transcriptase composition and the at least one oligonucleotide are provided in RT droplets.

7. The microfluidic method according to any one of claims 1 to 6, wherein: For a plurality of reservoirs, the at least one barcode sequence is attached to the surface of each reservoir of the plurality of reservoirs.

8. The microfluidic method of claim 7, wherein the at least one barcode sequence is released prior to transcription.

9. A microfluidic method for barcoding single-cell nucleic acids, the method comprising: - providing a microfluidic device comprising a chip, the chip comprising at least one microfluidic channel and a plurality of reservoirs, - injecting a carrier fluid into an inlet of the microfluidic channel, the carrier fluid comprising a plurality of first type droplets dispersed in the carrier fluid, wherein the first type droplets are 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 a single cell, wherein the single cell comprises a single-cell nucleic acid, - for a plurality of reservoirs, a first migration step, wherein at least one droplet of the first type in the plurality of droplets is moved into a reservoir of the plurality of reservoirs by buoyancy, - injecting a carrier fluid into an inlet of the microfluidic channel, the carrier fluid comprising a plurality of droplets of a second type dispersed in the carrier fluid, wherein the droplets of the second type are 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, - for a plurality of reservoirs, a second migration step, wherein at least a portion of at least one droplet of the second type enters a reservoir of the plurality of reservoirs, - for a plurality of reservoirs, said at least one droplet of said first type merges with said at least one droplet of said second type in each reservoir or at its edge, thereby producing a fused droplet, - and further comprising the following steps: a) for each fused droplet, hybridizing at least some of the single-cell nucleic acid to the at least one oligonucleotide in the fused droplet, b) in each fused droplet, reverse transcribing at least some of the single-cell nucleic acid present in the fused droplet, thereby producing single-cell cDNA, and c) attaching at least one barcode sequence to the single cell cDNA obtained in step b), wherein the at least one barcode sequence encodes the identity of the single cell, or - It also includes the following steps: a) for each fused droplet, hybridizing at least some of the single-cell nucleic acid from at least one cell to the at least one oligonucleotide in the fused droplet, wherein the at least one oligonucleotide comprises at least one barcode sequence, b) in each fused droplet, reverse transcribing at least some of the single-cell nucleic acids present in the fused droplet, thereby generating barcoded single-cell cDNA, wherein the at least one barcode sequence encodes the identity of the single cell.

10. The microfluidic method of claim 9, wherein the RT droplet further comprises at least one particle of a first type to which the at least one oligonucleotide is bound.

11. The microfluidic method of claim 9 or 10, wherein the RT droplets further comprise a polymer.

12. The microfluidic method according to any one of claims 9 to 11, wherein the plurality of droplets of the first type have an average volume of 1 pL to 3000 pL.

13. The microfluidic method according to any one of claims 9 to 12, wherein the plurality of droplets of the second type have an average volume of 10 pL to 5000 pL.

14. A microfluidic method according to any one of claims 9 to 13, wherein the first migration step of at least one droplet of the first type results in an occupation of 90% to 100% of the total number of reservoirs and / or results in a capture efficiency of 80% to 100% of the injected droplets of the first type.

15. The microfluidic method according to any one of claims 9 to 14, wherein the at least a portion of the at least one droplet of the second type refers to 90% to 100% of the total volume of the at least one droplet of the second type or refers to 10% to 30% of the total volume of the at least one droplet of the second type.

16. The microfluidic method according to any one of claims 9 to 15, wherein the fusing step results in a fusion efficiency between the first type of droplets and the second type of droplets of 90% to 100%.

17. A microfluidic device comprising a chip, wherein the chip comprises at least one microfluidic channel and a plurality of reservoirs, The microfluidic device further comprises an injection device for injecting a carrier fluid into an inlet of the microfluidic channel, the carrier fluid comprising a plurality of first type droplets dispersed in the carrier fluid, wherein the first type droplets are 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 a single cell, wherein the single cell comprises a nucleic acid from a single-cell transcriptome, - for a plurality of reservoirs, a first migration step, wherein at least one droplet of the first type in the plurality of droplets is moved into a reservoir of the plurality of reservoirs by buoyancy, The injection device is further used to inject a carrier fluid into the inlet of the microfluidic channel, the carrier fluid comprising a plurality of second type droplets dispersed in the carrier fluid, wherein the second type droplets are single-cell droplets or RT droplets, and wherein when the first type droplets are single-cell droplets, the second type droplets are RT droplets, or when the first type droplets are RT droplets, the second type droplets are single-cell droplets, - For multiple reservoirs, a second migration step, wherein, at least a portion of at least one droplet of the second type enters a reservoir of the plurality of reservoirs, each reservoir is used to perform a phenotypic assay on the at least one single cell, thereby mapping the phenotype of the at least one single cell for each reservoir, The microfluidic device further comprises a fusion device for merging the at least one droplet of the first type with the at least one droplet of the second type in each reservoir or at an edge thereof, thereby producing a fused droplet, in, a) for each fused droplet, hybridizing at least some of the nucleic acids from the single-cell transcriptome to the at least one oligonucleotide in the fused droplet, b) in each fused droplet, reverse transcribing at least some of the nucleic acids from the single-cell transcriptome present in the fused droplet, thereby generating single-cell cDNA, and c) attaching at least one barcode sequence to the single-cell cDNA obtained in step b), wherein the at least one barcode sequence encodes the identity of the single cell, or a) for each fused droplet, hybridizing at least some of the nucleic acids from the single-cell transcriptome from at least one cell in the fused droplet to the at least one oligonucleotide, wherein the at least one oligonucleotide comprises at least one barcode sequence, b) in each fused droplet, reverse transcribing at least some of the nucleic acids from the single-cell transcriptome present in the fused droplet, thereby generating barcoded single-cell cDNA, wherein the at least one barcode sequence encodes the identity of the single cell, - associating the identity of said single cell with said phenotype of said at least one single cell.

18. The microfluidic device of claim 17, wherein the injection device comprises a pressure controller or a syringe driver.

19. The microfluidic device of claim 17, wherein the fusion device is used to perform fusion in a selected area of ​​the chip.

20. The microfluidic device of claim 17, wherein the fusion device uses an electric field to perform fusion.

21. The microfluidic device of claim 17, wherein the at least one barcode sequence is attached to a surface of each reservoir of the plurality of reservoirs.

22. The microfluidic device of claim 17, wherein the at least one barcode sequence is released prior to transcription.

23. The microfluidic device of claim 17, wherein the plurality of droplets of the first type have an average volume of 1 pL to 3000 pL.

24. The microfluidic device of claim 17, wherein the plurality of droplets of the second type have an average volume of 10 pL to 5000 pL.

25. The microfluidic device of claim 17, wherein the first migration step of the at least one droplet of the first type results in an occupancy of 90% to 100% of the total reservoir and / or results in a capture efficiency of 80% to 100% of the injected droplets of the first type.

26. A microfluidic device according to claim 17, wherein the at least a portion of the at least one droplet of the second type refers to 90% to 100% of the total volume of the at least one droplet of the second type or refers to 10% to 30% of the total volume of the at least one droplet of the second type.

27. The microfluidic device of claim 17, wherein the fusing step results in a 90% to 100% fusion efficiency between the first type of droplets and the second type of droplets.

28. The microfluidic device of claim 17, wherein the microfluidic device further comprises a phenotypic assay device that performs a phenotypic assay on at least one single cell for each reservoir.

29. The microfluidic device of claim 28, wherein the phenotypic assay is the detection of a biological response.

30. The microfluidic device of claim 28, wherein the phenotypic assay comprises generating kinetic data.

31. The microfluidic device of claim 28, wherein the phenotypic assay comprises acquiring an image.

32. The microfluidic device according to claim 17, wherein the microfluidic device further comprises a cell sorter or a unit for generating droplets.

Images