Highly efficient compartmental loading of single cells

Abstract

Methods for producing compartmentalized single cell / barcoded bead compositions are provided. Aspects of the methods include contacting a composition of encapsulated single cells, e.g., double emulsion single cell droplets or gel-encapsulated single cells, with a plurality of microwells such that at least some of the microwells contain a single, deposited encapsulated cell (e.g., a single, deposited cell containing a double emulsion droplet or gel bead); releasing the single cells from their encapsulation, e.g., by disrupting the deposited double emulsion single cell droplets or dissolving the gel bead, to generate microwells containing the released single cells; and introducing barcoded beads into the microwells containing the released single cells to produce the compartmentalized single cell / barcoded bead composition. Compositions for carrying out the methods of the invention are also provided. The methods and compositions of the invention are useful for a variety of applications, such as single-cell sequencing applications.

Description

[Background technology]

[0001] Current technology allows for the measurement of gene expression in single cells in a massively parallel manner (e.g., >10,000 cells) by attaching cell-specific oligonucleotide barcodes to poly(A) mRNA molecules from individual cells as they colocalize with barcoding reagent beads within a compartment.

[0002] While variations and custom modifications are abundant in the published literature, the general workflow for scRNA-seq research can be summarized as follows. The first step in performing scRNA-seq is the isolation of viable single cells (or nuclei) from experimental samples, such as in vitro-grown cells, blood, or tissues of interest. Current methods then rely on isolating or compartmentalizing these single cells or their nuclei, along with barcoded oligonucleotides attached to beads, into physically separate compartments / divisions (e.g., microwells) or into individual droplets within a microfluidic device. For single-cell analysis, each compartment (e.g., microwell or droplet) typically contains one cell and one bead, with the oligonucleotides attached to the beads bearing the same unique, bead-specific (cell-specific) barcode. The isolated individual cells are then lysed to release mRNA molecules, which are then hybridized with barcoded oligo-dT primers attached to or released from the beads. After hybridization, the resulting oligo-dT-primed mRNA is converted into barcoded complementary DNA (cDNA) by reverse transcriptase. The barcoded cDNAs from different cells are then mixed together and amplified for follow-up expression analysis.

[0003] Early techniques capable of achieving massively parallel single-cell sequencing via miniaturized partitions were limited to a double Poisson distribution of cells and capture beads across the partition, with only a very small fraction of the partition population (typically 1%) containing both a single barcoded capture bead and a single cell.

[0004] More recently, key innovations in microfluidics and capture bead technology have enabled single-Poisson distribution across partitions (where only cells are Poisson loaded) in massively parallel single-cell sequencing. Examples of single-Poisson solutions include those offered by the Chromium (10x Genomics) system and the BD Rhapsody™ Single-Cell Analysis System (Becton Dickinson and Company). The BD Rhapsody™ Single-Cell Analysis System is a platform that enables high-throughput capture of nucleic acids from single cells using a simple cartridge workflow and multi-layer barcoding system. The resulting capture information can be used to generate various types of next-generation sequencing (NGS) libraries, including libraries suitable for whole-transcriptome analysis, e.g., targeted RNA analysis for discovery biology and highly sensitive transcript detection. Shum et al., "Quantitation of mRNA Transcripts and Proteins Using the BD Rhapsody" TM Single-Cell Analysis System”, Adv Exp Med Biol.2019;1129:63-79. Summary of the Invention

[0005] There is a need in the art to develop methods that provide better than Poisson loading of individual cells and beads within a compartment. Embodiments of the present invention allow for efficient (non-Poisson) loading of individual cells with a single bead into a compartment, including better than Poisson loading of individual cells and beads within a compartment.

[0006] Methods for producing compartmentalized single cell / barcoded bead compositions are provided. Aspects of the methods include contacting a composition of encapsulated single cells (e.g., double emulsion single cell droplets or gel-encapsulated single cells) with a plurality of microwells such that at least some of the microwells contain a single, deposited encapsulated cell (e.g., a single, deposited cell containing a double emulsion droplet or a gel bead); releasing the single cells from their encapsulation, e.g., by disrupting the deposited double emulsion single cell droplets or dissolving the gel beads, to generate microwells containing the released single cells; and introducing barcoded beads into the microwells containing the released single cells to produce the compartmentalized single cell / barcoded bead compositions. Compositions for carrying out the methods of the invention are also provided. The methods and compositions of the invention are used in a variety of applications, such as single-cell sequencing applications. [Brief explanation of the drawings]

[0007] The invention may be best understood from the following detailed description when read in conjunction with the accompanying drawings, in which:

[0008] [Figure 1] FIG. 1 illustrates the production of water-in-oil-in-water double emulsion droplets according to one embodiment of the present invention. [Figure 2A] FIG. 2A illustrates a schematic workflow according to one embodiment of the present invention. [Figure 2B] FIG. 2B illustrates a schematic workflow according to one embodiment of the present invention. Detailed Description of the Invention

[0009] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989).For the purposes of this disclosure, the following terms are defined below.

[0010] As used herein, the terms "associated" or "associated with" can mean that two or more species are identifiable as being in the same location at a time. Association can mean that two or more species are or were in similar containers. Association can be an informatics association. For example, digital information about two or more species can be stored and used to determine that one or more species were located in the same location at a time. Association can also be a physical association. In some embodiments, two or more associated species are "tethered," "attached," or "immobilized" to each other or to a common solid or semi-solid surface. Association can refer to covalent or non-covalent means for attaching a label to a solid or semi-solid support, such as a bead. Association can be a covalent bond between a target and a label. Association can include hybridization between two molecules (such as a target molecule and a label).

[0011] As used herein, the term "complementary" can refer to the ability for precise pairing between two nucleotides. For example, if a nucleotide at a given location in a nucleic acid can hydrogen bond with a nucleotide in another nucleic acid, the two nucleic acids are considered complementary to each other at that location. Complementarity between two single-stranded nucleic acid molecules can be "partial," in which only a portion of the nucleotides bind, or complete, in which complete complementarity exists between the single-stranded molecules. A first nucleotide sequence can be said to be the "complement" of a second sequence if the first nucleotide sequence is complementary to the second nucleotide sequence. A first nucleotide sequence can be said to be the "reverse complement" of a second sequence if the first nucleotide sequence is complementary to a sequence that is the reverse of the second sequence (i.e., the order of the nucleotides is reversed). As used herein, the terms "complement," "complementary," and "reverse complement" can be used interchangeably. From the present disclosure, it is understood that if a molecule can hybridize to another molecule, it can be the complement of the hybridized molecule.

[0012] As used herein, the term "nucleic acid" refers to a polynucleotide sequence or a fragment thereof. A nucleic acid can comprise nucleotides. A nucleic acid can be exogenous or endogenous to a cell. A nucleic acid can exist in a cell-free environment. A nucleic acid can be a gene or a fragment thereof. A nucleic acid can be DNA. A nucleic acid can be RNA. A nucleic acid can comprise one or more analogs (e.g., modified backbones, sugars, or nucleobases). Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, heterologous nucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to a sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queosine, and wiosine. "Nucleic acid," "polynucleotide," "target polynucleotide," and "target nucleic acid" can be used interchangeably.

[0013] Nucleic acids can contain one or more modifications (e.g., base modifications, backbone modifications) to provide the nucleic acid with new or enhanced characteristics (e.g., improved stability). Nucleic acids can contain nucleic acid affinity tags. Nucleosides can be base-sugar combinations. The base portion of a nucleoside can be a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. Nucleotides can be nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. In the case of nucleosides containing a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. In forming nucleic acids, the phosphate groups can covalently link adjacent nucleosides to each other to form a linear polymeric compound. The respective ends of this linear polymeric compound can then be further joined to form a circular compound. However, linear compounds are generally preferred. Furthermore, linear compounds can have internal nucleotide base complementarity and thus can fold to produce fully or partially double-stranded compounds. Within nucleic acids, the phosphate groups can be commonly referred to as forming the internucleoside backbone of the nucleic acid. The linkage or backbone can be a 3' to 5' phosphodiester bond.

[0014] Nucleic acids can contain modified backbones and / or modified internucleoside linkages. Modified backbones can include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Suitable modified nucleic acid backbones containing a phosphorus atom therein can include, for example, phosphorothioates, chiral phosphorothioates, phosphodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidate and aminoalkylphosphoramidate, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linkage analogs, and boranophosphates having reverse polarity where one or more internucleotide linkages are 3' to 3', 5' to 5', or 2' to 2' linkages.

[0015] Nucleic acids can contain polynucleotide backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These can include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, riboacetyl backbones, alkene-containing backbones, sulfamic acid backbones, methyleneimino and methylenehydrazino backbones, sulfonic acid and sulfonamide backbones, those with amide backbones, and others with mixed N, O, S, and CH2 constituent moieties.

[0016] Nucleic acids can include nucleic acid mimetics. The term "mimetics" is intended to include polynucleotides in which only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups; replacement of only the furanose ring can also be referred to as a sugar surrogate. The heterocyclic base moiety or modified heterocyclic base moiety can be maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid can be a peptide nucleic acid (PNA). In a PNA, the sugar backbone of a polynucleotide can be replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleotides can be retained and are directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone. The backbone in a PNA compound can contain two or more linked aminoethylglycine units, giving the PNA an amide-containing backbone. The heterocyclic base moiety is directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone.

[0017] The nucleic acid can include a morpholino backbone structure. For example, the nucleic acid can include a six-membered morpholino ring instead of a ribose ring. In some of these embodiments, phosphorodiamidates or other non-phosphodiester internucleoside linkages can replace the phosphodiester linkages.

[0018] Nucleic acids can contain linked morpholino units (e.g., morpholino nucleic acids) with heterocyclic bases attached to the morpholino ring. Linking groups can link the morpholino monomer units in morpholino nucleic acids. Nonionic morpholino-based oligomeric compounds may have fewer undesirable interactions with cellular proteins. Morpholino-based polynucleotides can be nonionic mimics of nucleic acids. Various compounds within the morpholino class can be conjugated using different linking groups. A further class of polynucleotide mimics can be called cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in nucleic acid molecules can be replaced with a cyclohexenyl ring. CeNA DMT-protected phosphoramidite monomers can be prepared and used to synthesize oligomeric compounds using phosphoramidite chemistry. Incorporation of CeNA monomers into nucleic acid chains can increase the stability of DNA / RNA hybrids. CeNA oligoadenylates can form complexes with nucleic acid complements with stability similar to that of the native complex. A further modification is Locked Nucleic Acids (LNAs), in which a 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring, thereby forming a 2'-C,4'-C-oxymethylene linkage, thereby forming a bicyclic sugar moiety. The linkage can be methylene (-CH), a group bridging the 2' oxygen atom and the 4' carbon atom (where n is 1 or 2). LNAs and LNA analogs can exhibit very high duplex thermal stability with complementary nucleic acids (Tm = +3 to +10°C), stability against 3'-exonuclease degradation, and good solubility properties.

[0019] Nucleic acids can also contain modifications or substitutions of nucleobases (often simply referred to as "bases"). As used herein, "unmodified" or "natural" nucleobases can include purine bases (e.g., adenine (A) and guanine (G)) and pyrimidine bases (e.g., thymine (T), cytosine (C), and uracil (U)). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine, and uracil. These may include tosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Modified nucleobases include tricyclic pyrimidines, such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G clamps, such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G clamps, such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidines (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidines (H-pyrido(3',2':4,5)pyrrolo[2,3-d]pyrimidin-2-one).

[0020] As used herein, the term "sample" can refer to a composition containing a target. Samples suitable for analysis by the disclosed methods, devices, and systems include cells, tissues, organs, or organisms. A cell sample is a composition composed of multiple cells, e.g., a composition containing multiple different cells, e.g., an aqueous composition of a single cell, and the number of cells can vary.

[0021] As used herein, the term "sampling device" or "device" can refer to a device that can collect a portion of a sample and / or place the portion on a substrate. A sample device can refer to, for example, a fluorescence-activated cell sorting (FACS) machine, a cell sorter, a biopsy needle, a biopsy device, a tissue sectioning device, a microfluidic device, a blade grid, and / or a microtome.

[0022] As used herein, the term "solid support" can refer to a discrete solid or semi-solid surface to which nucleic acids can be attached. A solid support can include any type of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar configuration made of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) to which nucleic acids can be immobilized (e.g., covalently or non-covalently). A solid support can be spherical (e.g., microsphere) or can include discrete particles that can have non-spherical or irregular shapes, such as cubes, rectangular prisms, pyramidal, cylindrical, conical, oval, or discoidal shapes. Beads can be non-spherical. A plurality of solid supports spaced apart in an array may not include a substrate. The term "solid support" can be used interchangeably with the term "bead."

[0023] As used herein, the term "target" can refer to a composition that can be analyzed according to embodiments of the present invention. Exemplary suitable targets for analysis by the disclosed methods, devices, and systems include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, and the like. Targets can be single-stranded or double-stranded. In some embodiments, targets can be proteins, peptides, or polypeptides. In some embodiments, targets are lipids. As used herein, "target" can be used interchangeably with "species."

[0024] As used herein, the term "reverse transcriptase" can refer to a group of enzymes with reverse transcriptase activity (i.e., catalyzing the synthesis of DNA from an RNA template). Generally, such enzymes include, but are not limited to, retroviral reverse transcriptases, retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retron reverse transcriptases, bacterial reverse transcriptases, group II intron-derived reverse transcriptases, and mutants, variants, or derivatives thereof. Non-retroviral reverse transcriptases include non-LTR retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retron reverse transcriptases, and group II intron reverse transcriptases. Examples of group II intron reverse transcriptases include Lactococcus lactis LI.LtrB intron reverse transcriptase, Thermosynechococcus elongatus TeI4c intron reverse transcriptase, or Geobacillus stearothermophilus GsI-IIC intron reverse transcriptase. Other classes of reverse transcriptases can include many classes of non-retroviral reverse transcriptases (ie, retrons, group II introns, and diversity-generating retroelements, among others).

[0025] As used herein, the term "gel" or "gel bead" can refer to a broad set of polymers that can reversibly form semi-solids, which can be considered hydrogels.

[0026] Detailed Description Methods for producing compartmentalized single cell / barcoded bead compositions are provided. Aspects of the methods include contacting a composition of encapsulated single cells, e.g., double emulsion single cell droplets or gel-encapsulated single cells, with a plurality of microwells such that at least some of the microwells contain a single, deposited encapsulated cell, e.g., a single, deposited cell containing a double emulsion droplet or gel bead; releasing the single cells from their encapsulation, e.g., by disrupting the deposited double emulsion single cell droplets or dissolving the gel beads, to generate microwells containing the released single cells; and introducing barcoded beads into the microwells containing the released single cells to produce the compartmentalized single cell / barcoded bead compositions. Compositions for carrying out the methods of the invention are also provided. The methods and compositions of the invention are useful for a variety of applications, such as single-cell sequencing applications.

[0027] Before describing the present invention in more detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0028] Where a range of values ​​is presented, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0029] Certain ranges are described herein by numerical values ​​preceded by the term "about." The term "about" is used herein to literally support the exact number it precedes, as well as a number that is near or approximately the number preceded by the term. In determining whether a number is near or approximately a specifically stated number, the unstated near or approximately number may be a number that, in the context in which it is presented, represents a substantial equivalent to the specifically stated number.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are now described.

[0031] All publications and patents cited herein are incorporated by reference to disclose and describe the methods and / or materials for which the publications are cited, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0032] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or the use of a "negative" limitation.

[0033] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual components and features which may be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0034] While the systems and methods are described for grammatical fluidity with functional descriptions, it is to be clearly understood that the claims should not be construed as necessarily limited by "means" or "step" limitation constructions unless expressly formulated under 35 U.S.C. § 112, but rather should be given the full scope of the meaning and equivalents of the definitions provided by the claims under the doctrine of equivalents, and that if a claim is expressly formulated under 35 U.S.C. § 112, then the full statutory equivalents under 35 U.S.C. § 112 should be given.

[0035] method As summarized above, methods for producing compartmentalized single cell / barcoded bead compositions are provided. By "compartmentalized single cell / barcoded bead composition" is meant a plurality of compositions (cell / bead compositions) each comprising a cell and a barcoded bead, wherein the cell / bead compositions are separated from one another by a physical barrier, e.g., one or more walls. Examples of compartments include, but are not limited to, wells, e.g., microwells of a microwell array, as described in more detail below. The number of compartmentalized single cell / barcoded bead compositions produced by the methods of the invention may vary, and in some cases, a plurality of compartmentalized single cell / barcoded bead compositions are produced, e.g., 1,000 to 1,000,000, 10 to 10,000,000 compartmentalized single cell / barcoded bead compositions are produced. As summarized above, aspects of the present method embodiments include contacting a composition of double emulsion droplets or gel-encapsulated single cells with a plurality of microwells such that at least a portion of the plurality of microwells contain single deposited cells containing double emulsion droplets or gel beads, disrupting the deposited double emulsion single cell droplets or dissolving the gel beads to generate microwells containing released single cells, and introducing barcoded beads to the microwells containing the released single cells to produce compartmentalized single cell / barcoded bead compositions. Aspects of the method are described in more detail below.

[0036] Encapsulated single cells As summarized above, aspects of the present methods include contacting a composition of encapsulated single cells (e.g., double emulsion single-cell droplets or gel-encapsulated single cells) with a plurality of microwells such that at least a portion of the plurality of microwells contain a single deposited encapsulated cell, e.g., a single deposited cell containing a double emulsion droplet or a gel bead. An encapsulated single cell refers to a cell that is completely surrounded by a material, e.g., a liquid or a solid, such that the cell is separated from its environment by the encapsulating material. Examples of encapsulated single cells that may be used in embodiments of the present invention include, but are not limited to, double emulsion single-cell droplets and gel-encapsulated single cells.

[0037] Double emulsion single cell droplets As summarized above, embodiments of the present method include contacting a double emulsion single-cell droplet composition with a plurality of microwells such that at least a portion of the plurality of microwells contain a single, stacked double emulsion single-cell droplet. Double emulsion droplets, such as those comprising an internal aqueous phase and an oil layer suspended in an external aqueous carrier phase, are known and find use in many industrial, medical, and research applications. Such applications may include, for example, drug delivery, cosmetic delivery vehicles, cell encapsulation, and synthetic biology. Compartmentalization of cells, chemicals, or molecules into millions of smaller compartments, such as may be achieved using double emulsion droplets, may isolate the reactions of each unit and allow for separate processing or analysis of each compartment. Double emulsion droplets used in embodiments of the present invention include an internal phase containing cells, an encapsulating layer of a second liquid phase immiscible with the internal liquid phase, and an external carrier liquid phase, which may comprise a liquid miscible with the internal phase. In some cases, the double emulsion single-cell droplet composition comprises an aqueous carrier phase within which resides a plurality of droplets, the plurality of droplets comprising a single cell residing in the inner aqueous phase encapsulated by an oil layer. In some cases, the double emulsion single-cell droplet composition comprises a water-in-oil-in-water double emulsion single-cell droplet. Because the double emulsion droplets used in embodiments of the present invention comprise cells in the inner liquid phase, e.g., the inner aqueous liquid phase, the double emulsion droplets are referred to herein as single-cell double emulsion droplets.

[0038] The parameters of the double emulsion droplets containing single cells of the double emulsion single-cell droplet composition used in embodiments of the present invention can be varied as desired. The diameter of the double emulsion droplets may vary in some cases from 1 to 1,000 μm, e.g., from 5 to 500 μm, including 10 to 100 μm; in some cases, the diameter is in the range of 20 to 50 μm, including 30 to 40 μm. In some cases, the double emulsion droplets are configured to have diameters corresponding to the microwells of a microwell array into which the droplets are compartmentalized, as described in more detail below. The double emulsion droplets of the composition can have a substantially uniform distribution of diameters, e.g., within a population of droplets for multiparameter evaluation, with a coefficient of variation (CV), which is the mean diameter relative to the standard deviation, of 10% or less, such as 5% or less, including 2.5% or less. Droplet diameter can be determined using a variety of techniques, including optical microscopy, laser light scattering, or other techniques. The double emulsion droplets of the compositions used in embodiments of the present invention may have varying internal volumes; in some cases, the double emulsion droplets have volumes ranging from 1 picoliter (pL) to 50 nanoliters (nL), for example, from 100 pL to 20 nL.

[0039] The double emulsion single-cell droplet compositions used in embodiments of the present invention can be prepared from any desired initial cell source. The cell source or sample used in the production of a given composition of double emulsion single-cell droplets can include a plurality of single cells. Cell samples can be derived from a variety of sources, including, but not limited to, tissue samples, biopsies, blood samples, cell cultures, and the like. Additionally, cell samples can be derived from specific organs, tissues, tumors, neoplasms, and the like. Furthermore, cells from any population can be the source of the cell sample used in the subject methods, such as populations of prokaryotic or eukaryotic cells, including, but not limited to, bacterial cells, plant cells, fungal cells, animal cells, e.g., mammalian cells such as human cells, rodent (e.g., mouse, rat, etc.) cells, insect cells, amphibian cells, yeast cells, and the like.

[0040] For example, as described herein, any method can be used to produce double emulsion droplets. In some cases, droplets can be generated using a microfluidic device in which an aqueous flow is used to prepare an aqueous-miscible core (sometimes called a "core") containing cells, which is then encapsulated in an immiscible oil shell (sometimes called a "shell") and an outer aqueous carrier phase. Figure 1 illustrates the production of water-in-oil-in-water double emulsion droplets. In some embodiments, the double emulsion droplets contain a stabilizer, such as a surfactant. Thus, the microdroplets can comprise surfactant-stabilized emulsions, e.g., surfactant-stabilized single emulsions or surfactant-stabilized double emulsions. Any convenient surfactant can be used. The surfactant used depends on many factors, such as the oil and aqueous phases (or other suitable immiscible phases, e.g., any suitable hydrophobic and hydrophilic phases) used in the emulsion. For example, when using aqueous droplets in fluorocarbon oil, the surfactant can have a hydrophilic block (PEG-PPO) and a hydrophobic fluorinated block (Krytox® FSH). However, for example, if the oil is replaced with a hydrocarbon oil, the surfactant is instead selected to have a hydrophobic hydrocarbon block, such as the surfactant ABIL EM90. Desirable properties that can be considered when selecting a surfactant include one or more of the following: (1) low viscosity; (2) immiscible with the polymer used to construct the device and therefore not swelling the device; (3) biocompatibility; (4) assay reagents do not dissolve in the surfactant; (5) the surfactant exhibits good gas solubility, allowing gases to enter and exit; (6) a boiling point of the surfactant higher than the temperature used in PCR (e.g., 95°C); (7) emulsion stability; (8) the surfactant stabilizes droplets of the desired size; (9) the surfactant is soluble in the carrier phase but not in the droplet phase; (10) the surfactant has limited fluorescent properties; and (11) the surfactant remains soluble in the carrier phase over a temperature range. Other surfactants, including ionic surfactants, can also be envisioned.Other additives may also be included to stabilize the microdroplets, including polymers that increase droplet stability at temperatures above 35° C. Further details may be found in U.S. Patent Application Publication No. 20170022538, the disclosure of which is incorporated herein by reference.

[0041] Further aspects of double emulsion droplets and methods for their preparation are provided in U.S. Patent Application Publication Nos. 20170022538, 20170121756, 20120211084, 201422035, and 2009131543; U.S. Patent Nos. 9,238,206, 8,802,027, 9,039,273, and 7,772,287; WO 2010104604, WO 2019110590, and WO 20200157269; and EP 11838713, the disclosures of which are incorporated herein by reference.

[0042] In some cases, producing a composition of double emulsion single cell droplets includes selecting double emulsion droplets of interest from an initial composition to produce a composition of double emulsion single cell droplets. Selecting refers to selecting or choosing double emulsion droplets of interest, e.g., droplets containing single cells, from an initial composition for further analysis; in some cases, the single cells may be cells of a particular type, etc. Selection as performed by embodiments of the present invention may result in a composition enriched for double emulsion droplets of interest, e.g., double emulsion droplets containing single cells. Selection in such embodiments may be performed using any convenient protocol. In some cases, selection includes the use of cell sorting, such as a fluorescence-activated cell sorting (FACS) protocol. Examples of cell sorters that may be used in such cases include the BD Biosciences FACSCalibur™ cell sorter, BD Biosciences FACSCount™ cell sorter, BD Biosciences FACSLyric™ cell sorter, BD Biosciences Via™ cell sorter, BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorter, BD Biosciences FACSAria™ II cell sorter, BD Biosciences FACSAria™ III cell sorter, BD Biosciences FACSAria™ fusion cell sorter, and BD Biosciences FACSMelody™ cell sorter, BD Biosciences FACSymphony™ S6 cell sorter, BD Biosciences FACSDiscover™ S8 cell sorter, etc. See also U.S. Patent Application Publication No. 20210261953. The disclosure of which is incorporated herein by reference.If desired, data on selected droplets may be obtained, for example, for QC purposes.

[0043] Gel-encapsulated single cells As summarized above, embodiments of the present methods include contacting a gel-encapsulated single cell composition with a plurality of microwells such that at least a portion of the plurality of microwells contain a single, deposited gel-encapsulated single cell. Gel-encapsulated single cells are single cells surrounded or encased within a gel material, such as a hydrogel material. The parameters of the gel-encapsulated single cells used in embodiments of the present invention can vary as desired. The diameter of the gel-encapsulated single cells may vary, in some cases, from 1 to 1,000 μm, e.g., from 5 to 500 μm, including 10 to 100 μm; in some cases, the diameter is in the range of 20 to 50 μm, including 30 to 40 μm. In some cases, the gel-encapsulated single cells are configured to have a diameter corresponding to a microwell of a microwell array into which the encapsulated single cells will be compartmentalized, as described in more detail below. Gel-encapsulated single cells of the composition may have a substantially uniform distribution of diameters, for example, within a droplet population for multiparameter evaluation, with the coefficient of variation (CV), which is the mean diameter relative to the standard deviation, being 10% or less, such as 5% or less, including 2.5% or less. Diameters can be determined using a variety of techniques, including optical microscopy, laser light scattering, or other techniques. Gel-encapsulated single cells used in embodiments of the present invention may have varying internal volumes; in some cases, double emulsion droplets have volumes ranging from 1 picoliter (pL) to 50 nanoliters (nL), e.g., 100 pL to 20 nL.

[0044] The compositions of gel-encapsulated single cells used in embodiments of the present invention can be prepared from any desired initial cell source. The cell source or sample used to produce a given composition of gel-encapsulated single cells can include multiple single cells. Cell samples can be derived from a variety of sources, including, but not limited to, tissue samples, biopsies, blood samples, cell cultures, and the like. Additionally, cell samples can be derived from specific organs, tissues, tumors, neoplasms, and the like. Furthermore, cells from any population can be the source of the cell sample used in the subject methods, such as populations of prokaryotic or eukaryotic cells, including, but not limited to, bacterial cells, plant cells, fungal cells, animal cells, e.g., mammalian cells such as human cells, rodent (e.g., mouse, rat, etc.) cells, insect cells, amphibian cells, yeast cells, and the like.

[0045] For example, any method may be used to fabricate gel-encapsulated single cells, as described herein. In some cases, single cells can be initially isolated in droplets. In some embodiments, single cells are encapsulated in droplets. In some embodiments, encapsulation of single cells in droplets is achieved using a microfluidic device including a droplet generator. For example, a population of single cells can flow through a channel of a microfluidic device, the microfluidic device including a droplet generator in fluid communication with the channel under conditions sufficient to cause inertial ordering of the cells within the channel, thereby providing periodic injection of cells into the droplet generator to encapsulate single cells within individual droplets. In some embodiments, a method of encapsulating single cells in droplets includes adding an immiscible phase fluid, e.g., oil, to generate an emulsion of droplets, each containing a single cell. Additional description of cell encapsulation using a microfluidic droplet generator can be found, for example, in U.S. Patent Application Publication No. 20150232942, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the droplets in which the single cells are encapsulated comprise a polymeric material. For example, suitable polymeric materials may include interpenetrating polymer networks (IPNs); synthetic hydrogels; semi-interpenetrating polymer networks (sIPNs); thermoresponsive polymers, etc. For example, in some embodiments, a suitable polymer includes a copolymer of polyacrylamide and poly(ethylene glycol) (PEG). In some embodiments, a suitable polymer includes a copolymer of polyacrylamide and PEG, further including acrylic acid. In some embodiments, the droplets encapsulating the single cells may be microgel droplets. In such embodiments, the microgel droplets may be hydrogel droplets comprising a hydrogel polymer.Suitable hydrogel polymers may include, but are not limited to, lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate (HEMA), ethyl methacrylate (EMA), propylene glycol methacrylate (PEMA), acrylamide (AAM), N-vinylpyrrolidone, methyl methacrylate (MMA), glycidyl methacrylate (GDMA), glycol methacrylate (GMA), ethylene glycol, fumaric acid, and the like. Some hydrogel polymers require the use of a crosslinker. Common crosslinkers include tetraethylene glycol dimethacrylate (TEGDMA) and N,N'-methylenebisacrylamide. Hydrogel droplets may be homopolymers or may contain copolymers of two or more of the aforementioned polymers. Exemplary hydrogel droplets include, but are not limited to, copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO); Pluronic™ F-127 (a bifunctional block copolymer of PEO and PPO of the nominal formula EO100-PO65-EO100, where EO is ethylene oxide and PO is propylene oxide); Poloxamer 407 (a triblock copolymer consisting of a central block of poly(propylene glycol) flanked by two hydrophilic blocks of poly(ethylene glycol); poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymer with a nominal molecular weight of 12,500 daltons and a PEO:PPO ratio of 2:1; poly(N-isopropylacrylamide)-based hydrogels (PNIPAAm-based hydrogels); PNIPAAm-acrylic acid copolymer (PNIPAAm-co-AAc); poly(2-hydroxyethyl methacrylate); poly(vinylpyrrolidone); and the like.

[0046] Further aspects of double emulsion droplets and methods for their preparation are described in U.S. Patent Application Publication No. 20220033893; WO 2022026243; and Shao et al., "Microfluidic Encapsulation of Single Cells by Alginate Microgels Using a Trigger-Gelled Strategy," Front Bioeng Biotechnol (published October 14, 2020); Mohajeri et al., "Cell encapsulation in alginate-based microgels using droplet microfluidics; a review on gelation methods and applications," Biomed Phys Eng Express. (2022) 8(2), PMID: 35073537; Zhang et al., "One-Step Generation and Purification of Cell-Encapsulated Hydrogel Microspheres With an Easily Assembled Microfluidic Device," Front Bioeng Biotechnol. (Published January 28, 2022) PMID: 35155414, the disclosure of which is incorporated herein by reference.

[0047] In some cases, producing a composition of double emulsion single-cell droplets includes selecting gel-encapsulated single cells of interest from an initial composition to produce a composition of gel-encapsulated single cells. Selection refers to selecting or choosing gel-encapsulated single cells of interest, e.g., gel-encapsulated single cells, from an initial composition of single-cell-containing inclusion bodies for further analysis; in some cases, the single cells may be cells of a particular type, etc. Selection as performed by embodiments of the present invention may result in a composition enriched for gel-encapsulated single cells of interest, e.g., gel-encapsulated single cells. Selection in such embodiments may be performed using any convenient protocol. In some cases, selection includes the use of cell sorting, such as a fluorescence-activated cell sorting (FACS) protocol. Examples of cell sorters that may be used in such cases include the BD Biosciences FACSCalibur™ cell sorter, BD Biosciences FACSCount™ cell sorter, BD Biosciences FACSLyric™ cell sorter, BD Biosciences Via™ cell sorter, BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorter, BD Biosciences FACSAria™ II cell sorter, BD Biosciences FACSAria™ III cell sorter, BD Biosciences FACSAria™ fusion cell sorter, and BD Biosciences FACSMelody™ cell sorter, BD Biosciences FACSymphony™ S6 cell sorter, BD Biosciences FACSDiscover™ S8 cell sorter, etc. See also U.S. Patent Application Publication No. 20210261953. The disclosure of which is incorporated herein by reference.If desired, data on selected droplets may be obtained, for example, for QC purposes.

[0048] Compartmentalization of encapsulated single cells into microwells Following production of a composition of encapsulated single cells, e.g., double emulsion single cell droplets or gel-encapsulated single cells, as described above, embodiments of the method include compartmentalizing the encapsulated single cells of the composition to produce compartmentalized encapsulated single cells. In some cases, compartmentalization involves distributing the encapsulated single cells into compartments such that multiple compartments contain a single encapsulated single cell, e.g., a single double emulsion droplet containing a cell or a single gel inclusion containing a single cell. "Compartmentalized" refers to the encapsulated single cells being disposed within a chamber or container that can define an at least partially fluidically isolated structure. At least partially fluidically isolated means that a given plurality of compartments can be separated from a plurality of other compartments by one or more fluid barriers, e.g., walls of solid material. In some cases, a compartment may be open to the environment at one location, e.g., an upper location, such that liquid can flow across the open portion of the compartment. For example, if the multiple compartments comprise a microwell array, the interiors of the given compartments are fluidically isolated from each other but are open to the environment at their upper ends, e.g., as described in more detail below. The compartments (e.g., chambers or containers such as microwells) can be defined by a solid material configured to house encapsulated single cells, e.g., double emulsion single-cell droplets or gel-encapsulated single cells.

[0049] In some embodiments, the plurality of sections is a plurality of microwells. The plurality of microwells may be randomly distributed across the substrate. In some embodiments, the plurality of microwells may be distributed across the substrate in an ordered pattern, e.g., an ordered array. In some embodiments, the plurality of microwells is distributed across the substrate in a random pattern, e.g., a random array. The microwells can be fabricated in a variety of shapes and sizes. Suitable well geometries include, but are not limited to, cylindrical, elliptical, cubic, conical, hemispherical, rectangular, or polyhedral, e.g., three-dimensional geometric shapes consisting of several planes, e.g., a rectangular prism, a hexagonal prism, an octagonal prism, an inverted triangular pyramid, an inverted square pyramid, an inverted pentagonal pyramid, an inverted hexagonal pyramid, or an inverted truncated pyramid. In some embodiments, non-cylindrical microwells, e.g., wells with an elliptical or square footprint, may offer advantages in that they can accommodate larger cells. In some embodiments, the upper and / or lower edges of the well walls may be rounded to avoid sharp corners, thereby reducing electrostatic forces that may arise due to electrostatic field concentration at sharp edges or points. Thus, the use of rounded corners can improve the ability to retrieve beads from the microwells. Microwell dimensions can be characterized in terms of absolute dimensions. In some cases, the average diameter of the microwells can range from about 5 μm to about 100 μm. In other embodiments, the average microwell diameter is at least 5 μm, at least 10 μm, at least 15 μm, at least 20 μm, at least 25 μm, at least 30 μm, at least 35 μm, at least 40 μm, at least 45 μm, at least 50 μm, at least 60 μm, at least 70 μm, at least 80 μm, at least 90 μm, or at least 100 μm. In still other embodiments, the average microwell diameter is at most 100 μm, at most 90 μm, at most 80 μm, at most 70 μm, at most 60 μm, at most 50 μm, at most 45 μm, at most 40 μm, at most 35 μm, at most 30 μm, at most 25 μm, at most 20 μm, at most 15 μm, at most 10 μm, or at most 5 μm.Of interest in certain embodiments are microwell diameters selected to correspond to the diameter of the encapsulated single cells, e.g., double emulsion single-cell droplets or gel-encapsulated single cells. The term "corresponding" in this context refers to a diameter selection that allows at most one droplet to enter the microwell. In some such cases, the microwell diameter exceeds the droplet diameter by 1 to 20 μm, e.g., 2 to 15 μm, including 3 to 10 μm. The volume of the microwells used in the methods of the present invention is, in some cases, approximately 200 μm. 3 ~about 800,000μm 3 In some embodiments, the microwell volume is at least 200 μm 3 , at least 500 μm 3 , at least 1,000 μm 3 , at least 10,000 μm 3 , at least 25,000 μm 3 , at least 50,000 μm 3 , at least 100,000 μm 3 , at least 200,000 μm 3 , at least 300,000 μm 3 , at least 400,000 μm 3 , at least 500,000 μm 3 , at least 600,000 μm 3 , at least 700,000 μm 3 Or at least 800,000 μm 3 In other embodiments, the microwell volume is at most 800,000 μm 3 , up to 700,000 μm 3 , up to 600,000 μm 3 , 500,000 μm 3 , up to 400,000 μm 3 , up to 300,000 μm 3 , up to 200,000 μm 3 , up to 100,000 μm 3 , up to 50,000 μm 3 , up to 25,000 μm 3 , up to 10,000 μm 3, up to 1,000 μm 3 , up to 500 μm 3 , or up to 200 μm 3 The number of microwells in a given device used in embodiments of the invention may vary, and in some cases the number is 100 or more, e.g., 250 or more, e.g., 500 or more, including 1000 or more, e.g., 5,000 or more, e.g., 10,000 or more, and in some cases the number is 15,000 or less, e.g., 12,500 or less. Microwells suitable for use in embodiments of the invention are further described in PCT Application Serial No. PCT / US2016 / 014612, published as WO 2016 / 118915, the disclosure of which is incorporated herein by reference. As used herein, a substrate can refer to a type of solid support. A substrate can, for example, include multiple microwells. For example, a substrate can be a well array including two or more microwells. In some embodiments, a microwell can include a small reaction chamber of a defined volume. In some embodiments, a microwell can capture one or more cells. In some embodiments, a microwell can capture only one cell. In some embodiments, a microwell can capture one or more solid supports. In some embodiments, a microwell can capture only one solid support. In some embodiments, a microwell captures a single cell and a single solid support (e.g., a bead).

[0050] When compartmentalizing encapsulated single-cell droplets, the encapsulated single cells can be placed into compartments, e.g., microwells, of a microwell array using any convenient protocol. A given composition of encapsulated single cells, e.g., double emulsion single-cell droplets or gel-encapsulated single cells, can be contacted with a structure, e.g., a microwell, to compartmentalize the gel-encapsulated single cells. Any convenient protocol can be used to compartmentalize the encapsulated single cells of the composition, e.g., dispensing, e.g., pipetting, placing the encapsulated single cells of the composition into compartments, flowing the composition over the surface of a well plate, etc. The encapsulated single-cell composition can contact multiple compartments, e.g., by gravity flow, which allows the encapsulated single cells to settle into the compartmentalization structure, e.g., the microwells. In some cases, the encapsulated single-cell composition can be contacted with an array of microwells, e.g., by flowing the encapsulated single-cell composition across the array of microwells, such that the encapsulated single cells pass through the openings of the microwells and are deposited within the microwells. The composition containing the encapsulated single cells may flow through a flow cell in fluid communication with the microwells. When the composition of encapsulated single cells flows across the openings of the microwells of a microwell array, for example, the flow rate of the composition may vary in some cases from 1 μl / s to 5 ml / s, e.g., 18 μl / s to 300 μl / s. Suitable protocols and systems for compartmentalizing double emulsion droplets into microwells are described in PCT Application No. PCT / US2016 / 014612, published as WO 2016 / 118915, the disclosure of which is incorporated herein by reference.

[0051] As summarized above, in some embodiments, the encapsulated single cell composition contacts a plurality of microwells such that at least a portion of the plurality of microwells contain a single (i.e., single) deposited encapsulated single cell. The number of microwells containing a deposited encapsulated single cell can vary, but in some cases, a majority of the plurality of microwells contain a single deposited encapsulated single cell, e.g., a double emulsion single-cell droplet or a gel-encapsulated single cell. In some cases, 75% or more, e.g., 90% or more (including substantially all) of the microwells of the array can contain a deposited encapsulated single cell.

[0052] After contacting the encapsulated single cell composition with the plurality of microwells so that at least a portion of the plurality of microwells contain a single, deposited encapsulated single cell, e.g., as described above, the resulting plurality of compartments may be evaluated, if desired, to assess how many compartments contain encapsulated single cells, e.g., how many compartments contain one (i.e., a single) encapsulated single cell. If performed, such evaluation may be performed using a variety of different protocols. In some cases, the evaluating may include imaging the plurality of compartments. The compartmentalized encapsulated single cells may be imaged using any convenient protocol to obtain image data of the compartmentalized encapsulated single cells. The image data acquired may vary. Image data may be acquired for any droplet of interest, acquired from a compartment containing an encapsulated single cell of interest. The type of image data acquired may vary and may include live cell image data. Any convenient protocol can be used to obtain image data of the droplets within the partitions. Examples of imaging protocols that can be used include, but are not limited to, microscopic imaging protocols such as phase contrast microscopy, fluorescence microscopy, quantitative phase contrast microscopy, and holotomography. Images can be generated, for example, by fluorescence imaging. Imaging can include microscopy techniques such as bright-field imaging, oblique illumination, dark-field imaging, dispersion staining, phase contrast, differential interference contrast, interference reflection microscopy, fluorescence, confocal, and single-plane illumination, or any combination thereof. Imaging can include imaging a portion of a sample (e.g., a slide / array). Imaging can include imaging at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the partitioned droplets. In some cases, imaging can be performed in discrete steps (e.g., the images may not be consecutive). Imaging can include taking at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different images.The imaging can include taking up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different images. Optionally, the image data can include images taken from two or more separate imaging iterations, each of which includes a labeling step followed by an imaging step. In such cases, the acquisition of image data from the sectioned cells can be considered a cyclic imaging step. In some cases, the imaging is by bright-field and fluorescent imaging using, for example, a BD Rhapsody™ scanner (Becton Dickinson and Company).

[0053] Destruction of encapsulated single cells After contact and any desired evaluation, the deposited encapsulated single cells, such as double emulsion single-cell droplets or gel-encapsulated single cells, can be disrupted to generate microwells containing released single cells, e.g., as described above. Any convenient protocol can be used to disrupt the double emulsion droplets to release their single-cell cargo. In some cases, disruption of the double emulsion droplets is achieved by contacting the deposited double emulsion single-cell droplets with a disrupting agent to release the droplets' cells. In such cases, any convenient disrupting agent that disrupts the double emulsion droplets to release the single cells can be used. In some cases, the double emulsion droplets can be disrupted by contacting the droplets with an alternating electric field or a chemical agent. Examples of chemical disrupting agents that can be used include, but are not limited to, anionic surfactants, sodium dodecyl sulfate (SDS), and the like. If a chemical disrupting agent is used, the chemical disrupting agent can be contacted with the droplet using any convenient method, such as by flowing a liquid of the chemical disrupting agent, e.g., an aqueous composition, across the openings of the multiple sections, as described above. In still other cases, a change in pressure can be used as the disrupting agent. Thus, any convenient protocol can be used as desired, including, but not limited to, enzymatic activity, external pressure, alternating current, or dielectric / magnetic fields. If desired, the gel-encapsulated single cells can be disrupted using, for example, any convenient protocol.

[0054] After disruption of encapsulated single cells, such as double emulsion droplets, the cells within the compartments are released, and the released cells can be washed, if desired. Any convenient washing protocol can be implemented. For example, to wash the released cells, a suitable washing solution, such as a buffer solution, can be flowed across the openings of the multiple compartments, as described above. Suitable washing solutions include, but are not limited to, phosphate-buffered saline (PBS). Different modes of fluid flow control can be utilized at different points in the assay procedure; for example, forward flow (relative to the inlet and outlet of a given microwell chamber), reverse flow, oscillating or pulsatile flow, or combinations thereof can all be used. In some embodiments, oscillating or pulsatile flow can be applied during assay wash / rinse steps to facilitate complete and efficient exchange of fluid within one or more microwell flow cells or chambers.

[0055] Introducing barcoded beads Embodiments of the present method further include introducing barcoded beads into microwells containing single cells to produce a compartmented single-cell / barcoded bead composition. Accordingly, embodiments of the present method include providing beads (or similar particle structures) having surface-bound barcode nucleic acids to compartments containing single cells, where the bound barcode nucleic acids are used in preparing nucleic acid sequence-ready compositions, e.g., sequence-ready libraries, from the compartmented cells. In some cases, the bead-bound barcode nucleic acids (i.e., the barcode nucleic acids of the barcoded beads) include a target binding region that binds to a complementary sequence in a nucleic acid species of interest in the cell. For example, if the target nucleic acid species is cellular mRNA, the bead-bound barcode nucleic acid may include a poly(T) domain as the target binding region. In addition to the target binding region, the bound nucleic acid may further include one or more additional domains, such as, but not limited to, a cell labeling domain, a barcode domain, a molecular index domain (e.g., a unique molecular identifier (UMI) domain), a universal primer binding domain, etc. Further details regarding barcoded beads (or similar barcoded particles) having bound barcode nucleic acids that can be provided within the compartments of embodiments of the present invention can be found in U.S. Patent Application Publication No. 2018 / 0088112; U.S. Patent Application Publication No. 2018 / 0200710; U.S. Patent Application Publication No. 2018 / 0346970; U.S. Patent Application Publication No. 2019 / 0056415; U.S. Patent Application Publication No. 2020 / 0248263; U.S. Patent Application Publication No. 2020 / 0299672; and U.S. Patent Application Publication No. 2021 / 0171940, the disclosures of which are incorporated herein by reference. Barcoded beads can be introduced into multiple compartments using any convenient protocol. For example, barcoded beads can be introduced into the compartments by gravity flow, allowing the beads to settle into compartmentalized structures, such as microwells. In some cases, the liquid composition of beads contacts the array of microwells such that the beads are deposited in the microwells, e.g., by flowing the composition of beads across the array of microwells such that the beads are deposited in the microwells.The composition comprising the beads may flow through a flow cell in fluid communication with the microwells. When the bead composition flows across the openings of the microwells of a microwell array, for example, the flow rate of the composition may vary in some cases from 1 μl / s to 5 ml / s, e.g., 18 μl / s to 300 μl / s. The beads with bound nucleic acids may be provided within the compartments using any convenient protocol, including, but not limited to, those described above for compartmentalizing cells and further described in PCT Application No. PCT / US2016 / 014612, published as WO 2016 / 118915, the disclosure of which is incorporated herein by reference.

[0056] Although the above aspects of the invention are described with respect to introducing droplets, disrupting the droplets, and then introducing beads into the compartment, the invention is not so limited. For example, the order of the above steps can be changed as needed. For example, beads can be compartmentalized into cells before or after the droplets, or in some cases in combination with the droplets, as desired. In some cases, particles, e.g., beads, are provided to the compartment after the release of cells from the droplets. In some cases, particles, e.g., beads, are provided to the compartment before the release of cells from the droplets.

[0057] After introducing barcoded beads into the microwells, for example, as described above, the resulting microwells can be evaluated, if desired, to assess, for example, how many compartments contain cells and beads. If performed, such evaluation can be performed using a variety of different protocols. In some cases, evaluating can include imaging multiple compartments. The compartmented droplets can be imaged using any convenient protocol to obtain image data of the compartmented droplets, for example, as described above.

[0058] For example, a compartmentalized single cell / barcoded bead composition prepared as described above includes cells spatially adjacent to barcoded beads, i.e., beads (or similar particles) to which barcode nucleic acids containing target binding regions, e.g., as described above, are attached. When the barcode nucleic acid is adjacent to a target in a single cell, the target can hybridize to the target binding domain of the barcode nucleic acid. If desired, the barcode nucleic acids can be contacted in a non-depletable ratio so that each distinct target can associate with a barcode nucleic acid with its own unique UMI.

[0059] Further processing Following cell and bead partitioning, the cells can be lysed to release the target molecule, as described above, so that the released target molecule, e.g., nucleic acid, can bind to the target-binding region of the barcode nucleic acid to produce the captured nucleic acid. Cell lysis can be achieved by a variety of means, such as chemical or biochemical means, osmotic shock, or thermal, mechanical, or optical lysis. Particles can be lysed by adding a cell lysis buffer containing detergent (e.g., SDS, Li dodecyl sulfate, Triton X-100, Tween-20, or NP-40), organic solvent (e.g., methanol or acetone), or digestive enzymes (e.g., proteinase K, pepsin, or trypsin), or any combination thereof. To increase target-to-barcode association, the diffusion rate of the target molecule can be altered, for example, by lowering the temperature and / or increasing the viscosity of the lysate. In some embodiments, the sample can be lysed using filter paper. The filter paper can be immersed top-side in the lysis buffer. The filter paper can be applied to the sample with pressure that can facilitate sample lysis and hybridization of the sample's targets to the substrate. In some embodiments, lysis can be performed by mechanical lysis, thermal lysis, optical lysis, and / or chemical lysis. Chemical lysis can include the use of digestive enzymes such as proteinase K, pepsin, and trypsin. Lysis can be performed by adding a lysis buffer to the substrate. The lysis buffer can include Tris HCl. The lysis buffer can include at least about 0.01, 0.05, 0.1, 0.5, or 1 M or more of Tris HCl. The lysis buffer can include up to about 0.01, 0.05, 0.1, 0.5, or 1 M or more of Tris HCl. The lysis buffer can include about 0.1 M Tris HCl. The pH of the lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. The pH of the lysis buffer can be up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or higher.In some embodiments, the pH of the lysis buffer is about 7.5. The lysis buffer can include a salt (e.g., LiCl). The concentration of the salt in the lysis buffer can be at least about 0.1, 0.5, or 1 M or more. The concentration of the salt in the lysis buffer can be up to about 0.1, 0.5, or 1 M or more. In some embodiments, the concentration of the salt in the lysis buffer is about 0.5 M. The lysis buffer can include a detergent (e.g., SDS, Li dodecyl sulfate, triton X, Tween, NP-40). The concentration of the detergent in the lysis buffer can be at least about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7% or more. The concentration of detergent in the lysis buffer can be up to about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7% or more. In some embodiments, the concentration of detergent in the lysis buffer is about 1% Li-dodecyl sulfate. The time used in the lysis method can depend on the amount of detergent used. In some embodiments, the more detergent used, the shorter the time required for lysis. The lysis buffer can include a chelating agent (e.g., EDTA, EGTA). The concentration of the chelating agent in the lysis buffer can be at least about 1, 5, 10, 15, 20, 25, or 30 mM or more. The concentration of the chelating agent in the lysis buffer can be up to about 1, 5, 10, 15, 20, 25, or 30 mM or more. In some embodiments, the concentration of the chelating agent in the lysis buffer is about 10 mM. The lysis buffer can include a reducing reagent (e.g., beta-mercaptoethanol, DTT). The concentration of the reducing reagent in the lysis buffer can be at least about 1, 5, 10, 15, or 20 mM or more. The concentration of the reducing reagent in the lysis buffer can be up to about 1, 5, 10, 15, or 20 mM or more. In some embodiments, the concentration of the reducing reagent in the lysis buffer is about 5 mM.In some embodiments, the lysis buffer can comprise about 0.1 M TrisHCl, about pH 7.5, about 0.5 M LiCl, about 1% lithium dodecyl sulfate, about 10 mM EDTA, and about 5 mM DTT. Lysis can be performed at a temperature of about 4, 10, 15, 20, 25, or 30° C. Lysis can be performed for about 1, 5, 10, 15, or 20 minutes or more. Lysed cells can comprise at least about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, or 700,000 or more target nucleic acid molecules. Lysed cells can comprise up to about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, or 700,000 or more target nucleic acid molecules.

[0060] After cell lysis and release of nucleic acid molecules therefrom, the nucleic acid molecules can randomly associate with the barcode nucleic acid of a co-localized solid support, e.g., a bead. Association can involve hybridization of the target recognition region of the barcode nucleic acid with a complementary portion of the target nucleic acid molecule (e.g., the oligo(dT) of the barcode can interact with the poly(A) tail of the target). Assay conditions (e.g., buffer pH, ionic strength, temperature, etc.) used for hybridization can be selected to promote the formation of specific, stable hybrids. In some embodiments, nucleic acid molecules released from lysed cells can associate with (e.g., hybridize to) multiple probes on a substrate. If the probes contain oligo(dT), mRNA molecules can hybridize to the probes and be reverse transcribed. The oligo(dT) portion of the oligonucleotide can act as a primer for first-strand synthesis of cDNA molecules, for example, when subjected to DNA synthesis reaction conditions to produce first-strand cDNA domains containing the capture nucleic acid.

[0061] If desired, a given workflow can include a pooling step in which a product composition, e.g., comprised of captured nucleic acids, synthesized first-strand cDNA, or synthesized second-strand cDNA, is combined or pooled with product compositions obtained from one or more additional samples, e.g., cells. In some cases, the pooling step is performed immediately after a hybridization step between the barcode nucleic acid and the target nucleic acid, e.g., as outlined above. The number of different product compositions produced from different samples, e.g., cells, that are combined or pooled in such embodiments can vary, and in some cases the number can range from 2 to 100,000,000, e.g., 2 to 10,000,000, e.g., 2 to 1,000,000, e.g., 3 to 200,000, including 4 to 100,000, e.g., 5 to 50,000, and in some cases the number can range from 100 to 10,000, e.g., 1,000 to 5,000. Before or after pooling, the product composition(s) can be amplified, for example, by polymerase chain reaction (PCR), as described in more detail below. Once the target-barcode nucleic acid molecules are pooled, all further processing can proceed within a single reaction vessel. Further processing can include, for example, reverse transcription reactions, amplification reactions, cleavage reactions, dissociation reactions, and / or nucleic acid extension reactions. Further processing reactions can be performed within microwells, i.e., without first pooling the labeled target nucleic acid molecules from multiple cells.

[0062] The present disclosure provides methods for generating target-barcode nucleic acid conjugates using any convenient protocol, such as reverse transcription or nucleotide extension. The target-barcode conjugates can contain complementary sequences of all or part of the barcode and target nucleic acids. Reverse transcription of the associated RNA molecules can occur by adding a reverse transcription primer along with a reverse transcriptase. The reverse transcription primer can be an oligo(dT) primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. The oligo(dT) primer can be 12 to 18 nucleotides long and binds to the endogenous poly(A) tail at the 3' end of mammalian mRNA. The random hexanucleotide primer can bind to the mRNA at various complementary sites. The target-specific oligonucleotide primer typically selectively primes the mRNA of interest. Reverse transcription can occur repeatedly to produce multiple cDNA molecules. The methods disclosed herein can include performing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 reverse transcription reactions. The methods can include performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.

[0063] One or more nucleic acid amplification reactions can be performed to generate multiple copies of a target nucleic acid molecule. Amplification can be performed in a multiplexed manner, where multiple target nucleic acid sequences are amplified simultaneously. The amplification reaction can be used to add sequencing adapters to the nucleic acid molecule. The amplification reaction can include amplifying at least a portion of the sample label, if present. The amplification reaction can include amplifying at least a portion of the cell label and / or barcode sequence (e.g., molecular label). The amplification reaction can include amplifying at least a portion of the sample tag, cell label, spatial label, barcode sequence (e.g., molecular label), target nucleic acid, or a combination thereof. The amplification reaction can include amplifying 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100%, or a range or number between any two of these values ​​of the plurality of nucleic acids. The method can further include performing one or more cDNA synthesis reactions to produce one or more cDNA copies of the target barcode molecule comprising the sample label, cell label, spatial label, and / or barcode sequence (e.g., molecular label).

[0064] In some embodiments, amplification can be performed using polymerase chain reaction (PCR). As used herein, PCR can refer to a reaction for in vitro amplification of specific DNA sequences by simultaneous primer extension of complementary strands of DNA. As used herein, PCR can encompass derivative forms of the reaction, including, but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, digital PCR, and assembly PCR.

[0065] Nucleic acid amplification can include non-PCR-based methods. Examples of non-PCR-based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or intercircle amplification. Other non-PCR-based amplification methods include multiple cycles of DNA-dependent RNA polymerase-driven RNA transcription amplification or RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR) and Qβ replicase (Qβ) methods, the use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cleaved before extension and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5' exonuclease activity, rolling circle amplification, and branch extension amplification (RAM). In some embodiments, amplification does not produce circularized transcripts.

[0066] In some embodiments, the methods disclosed herein further include performing a polymerase chain reaction on the nucleic acid (e.g., RNA, DNA, cDNA) to produce a labeled amplicon (e.g., a stochastically labeled amplicon). The labeled amplicon can be a double-stranded molecule. The double-stranded molecule can include a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized to a DNA molecule. One or both strands of the double-stranded molecule can include a sample label, a spatial label, a cell label, and / or a barcode sequence (e.g., a molecular label). The labeled amplicon can be a single-stranded molecule. The single-stranded molecule can include DNA, RNA, or a combination thereof. The nucleic acid of the present disclosure can include a synthetic nucleic acid or a modified nucleic acid. Thus, the method can include producing an amplicon composition from a first-strand cDNA domain containing a capture nucleic acid.

[0067] Amplification can include the use of one or more non-natural nucleotides. Non-natural nucleotides can include photolabile or triggerable nucleotides. Examples of non-natural nucleotides include, but are not limited to, peptide nucleic acids (PNAs), morpholinos and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs). Non-natural nucleotides can be added in one or more cycles of the amplification reaction. The addition of non-natural nucleotides can be used to identify products at specific cycles or time points in the amplification reaction.

[0068] Performing one or more amplification reactions can include the use of one or more primers. The one or more primers can include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one or more primers can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one or more primers can include 12 to fewer than 15 nucleotides. The one or more primers can anneal to at least a portion of the multiple labeled targets (e.g., stochastically labeled targets). The one or more primers can anneal to the 3' or 5' ends of the multiple labeled targets. The one or more primers can anneal to an internal region of the multiple labeled targets. The internal region can be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, or 1000 nucleotides from the 3' end of the multiple labeled targets. The one or more primers can comprise a fixed panel of primers. The one or more primers can comprise at least one or more custom primers. The one or more primers may include at least one or more control primers. The one or more primers may include at least one or more gene-specific primers.

[0069] The one or more primers can include a universal primer. The universal primer can anneal to the universal primer binding site. The one or more custom primers can anneal to a first sample label, a second sample label, a spatial label, a cell label, a barcode sequence (e.g., a molecular label), a target, or any combination thereof. The one or more primers can include a universal primer and a custom primer. The custom primers can be designed to amplify one or more targets. The targets can include a subset of all nucleic acids in one or more samples. The targets can include a subset of all labeled targets in one or more samples. The one or more primers can include at least 96 or more custom primers. The one or more primers can include at least 960 or more custom primers. The one or more primers can include at least 9600 or more custom primers. The one or more custom primers can anneal to two or more different labeled nucleic acids. The two or more different labeled nucleic acids can correspond to one or more genes.

[0070] Any amplification scheme can be used in the disclosed methods. For example, in one scheme, the first round of PCR can amplify molecules attached to beads using a gene-specific primer and a primer for the universal Illumina sequencing primer 1 sequence. The second round of PCR can amplify the first PCR product using a nested gene-specific primer adjacent to the Illumina sequencing primer 2 sequence and a primer for the universal Illumina sequencing primer 1 sequence. The third round of PCR adds P5 and P7 and a sample index to convert the PCR products into an Illumina sequencing library. Sequencing using 150 bp x 2 sequencing can reveal cell label and barcode sequences (e.g., molecular labels) on Read 1, genes on Read 2, and sample indexes on Index 1 read.

[0071] In some embodiments, nucleic acids can be removed from a substrate using chemical cleavage. For example, chemical groups or modified bases present in the nucleic acid can be used to facilitate its removal from the solid support. For example, enzymes can be used to remove nucleic acids from a substrate. For example, nucleic acids can be removed from a substrate by restriction endonuclease digestion. For example, nucleic acids containing dUTP or ddUTP can be removed from a substrate using treatment with uracil-d-glycosylase (UDG). For example, enzymes that remove nucleotides, such as base excision repair enzymes, e.g., apurinic / apyrimidinic (AP) endonucleases, can be used to remove nucleic acids from a substrate. In some embodiments, nucleic acids can be removed from a substrate using photocleavable groups and light. In some embodiments, nucleic acids can be removed from a substrate using a cleavable linker. For example, the cleavable linker can include at least one of biotin / avidin, biotin / streptavidin, biotin / neutravidin, Ig-Protein A, a photolabile linker, an acid- or base-labile linker group, or an aptamer.

[0072] In some embodiments, amplification can be performed on a substrate using, for example, bridge amplification. The cDNA can be homopolymer-tailed to generate compatible ends for bridge amplification using oligo(dT) probes on the substrate. In bridge amplification, a primer complementary to the 3' end of the template nucleic acid can be the first primer of each pair covalently attached to solid particles. A sample containing the template nucleic acid is contacted with the particles and subjected to a single thermal cycle, allowing the template molecule to anneal to the first primer, which then extends in the forward direction by adding nucleotides to form a duplex molecule consisting of the template molecule and a newly formed DNA strand complementary to the template. The heating step of the next cycle denatures the duplex molecule, releasing the template molecule from the particle and leaving the complementary DNA strand attached to the particle via the first primer. During the annealing phase of the subsequent annealing and extension step, the complementary strand can hybridize to a second primer complementary to the segment of the complementary strand at the position removed from the first primer. This hybridization allows the complementary strand to be covalently fixed to the first primer and to form a bridge between the first and second primers, which are fixed to the second primer by hybridization. During the extension step, the second primer can be extended in the opposite direction by the addition of nucleotides in the same reaction mixture, thereby converting the bridge into a double-stranded bridge. The next cycle then begins, denaturing the double-stranded bridge to yield two single-stranded nucleic acid molecules, each with one end attached to the particle surface via the first and second primers and the other end unattached. During the annealing and extension step of this second cycle, each strand can hybridize to additional, previously unused, complementary primers on the same particle to form a new single-stranded bridge. The two previously unused hybridized primers then extend, converting the two new bridges into double-stranded bridges.The amplification reaction may include amplifying at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 100% of the plurality of nucleic acids.

[0073] Amplification of the labeled nucleic acid can include PCR-based or non-PCR-based methods. Amplification of the labeled nucleic acid can include exponential amplification of the labeled nucleic acid. Amplification of the labeled nucleic acid can include linear amplification of the labeled nucleic acid. Amplification can be performed by polymerase chain reaction (PCR). PCR can refer to a reaction for in vitro amplification of specific DNA sequences by simultaneous primer extension of complementary strands of DNA. PCR can encompass derivative forms of the reaction, including, but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, digital PCR, suppression PCR, semi-suppression PCR, and assembly PCR.

[0074] In some embodiments, amplification of the labeled nucleic acid comprises a non-PCR-based method. Examples of non-PCR-based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or intercircle amplification. Other non-PCR-based amplification methods include multiple cycles of DNA-dependent RNA polymerase-driven RNA transcription amplification or RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR), Qβ replicase (Qβ), the use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cleaved prior to extension and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5' exonuclease activity, rolling circle amplification, and / or branch extension amplification (RAM).

[0075] In some embodiments, the methods disclosed herein further include performing a nested polymerase chain reaction on the amplified amplicon (e.g., target). The amplicon can be a double-stranded molecule. The double-stranded molecule can include a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized to a DNA molecule. One or both strands of the double-stranded molecule can include a sample tag or molecular identifier label. Alternatively, the amplicon can be a single-stranded molecule. The single-stranded molecule can include DNA, RNA, or a combination thereof. The nucleic acids of the present invention can include synthetic or modified nucleic acids.

[0076] In some embodiments, the methods involve repeatedly amplifying labeled nucleic acids to produce multiple amplicons. The methods disclosed herein can include performing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amplification reactions. Alternatively, the methods can include performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amplification reactions.

[0077] The amplification can further include adding one or more control nucleic acids to one or more samples containing the plurality of nucleic acids. The amplification can further include adding one or more control nucleic acids to the plurality of nucleic acids. The control nucleic acids can include a control label.

[0078] Amplification can include the use of one or more non-natural nucleotides. Non-natural nucleotides can include photolabile and / or triggerable nucleotides. Examples of non-natural nucleotides include, but are not limited to, peptide nucleic acids (PNAs), morpholinos and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs). Non-natural nucleotides can be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides can be used to identify products at specific cycles or time points in the amplification reaction.

[0079] Performing one or more amplification reactions can include using one or more primers. The one or more primers can include one or more oligonucleotides. The one or more oligonucleotides can include at least about 7 to 9 nucleotides. The one or more oligonucleotides can include 12 to less than 15 nucleotides. The one or more primers can anneal to at least a portion of the multiple labeled nucleic acids. The one or more primers can anneal to the 3' and / or 5' ends of the multiple labeled nucleic acids. The one or more primers can anneal to an internal region of the multiple labeled nucleic acids. The internal region can be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, or 1000 nucleotides from the 3' end of the plurality of labeled nucleic acids. The one or more primers can comprise a fixed panel of primers. The one or more primers can comprise at least one or more custom primers. The one or more primers can include at least one or more control primers. The one or more primers can include at least one or more housekeeping gene primers. The one or more primers can include a universal primer. The universal primer can anneal to a universal primer binding site. The one or more custom primers can anneal to a first sample tag, a second sample tag, a molecular identifier label, a nucleic acid, or a product thereof. The one or more primers can include a universal primer and a custom primer. The custom primer can be designed to amplify one or more target nucleic acids. The target nucleic acids can include a subset of the total nucleic acids in one or more samples. In some embodiments, the primers are probes attached to the array of the present disclosure.

[0080] In some embodiments, barcoding (e.g., stochastically barcoding) a plurality of targets in a sample further includes generating an indexed library of barcoded targets (e.g., stochastically barcoded targets) or barcoded target fragments. The barcode sequences of different barcodes (e.g., molecular labels of different stochastic barcodes) can be different from each other. Generating an indexed library of barcoded targets includes generating a plurality of indexed polynucleotides from the plurality of targets in the sample. For example, in the case of an indexed library of barcoded targets including a first indexed target and a second indexed target, the labeled region of the first indexed polynucleotide can differ from the labeled region of the second indexed polynucleotide by about, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or a number or range between any two of these values. In some embodiments, generating an indexed library of barcoded targets includes contacting a plurality of targets, e.g., mRNA molecules, with a plurality of oligonucleotides comprising a poly(T) region and a label region, and performing first-strand synthesis using a reverse transcriptase to produce single-stranded, labeled cDNA molecules, each comprising a cDNA region and a label region, wherein the plurality of targets comprises at least two mRNA molecules of different sequences, and the plurality of oligonucleotides comprises at least two oligonucleotides of different sequences. Generating an indexed library of barcoded targets can further include amplifying the single-stranded, labeled cDNA molecules to produce double-stranded, labeled cDNA molecules, and performing nested PCR on the double-stranded, labeled cDNA molecules to produce labeled amplicons. In some embodiments, the method can include generating adapter-labeled amplicons.

[0081] Barcoding (e.g., stochastic barcoding) can involve using nucleic acid barcodes or tags to label individual nucleic acid (e.g., DNA or RNA) molecules. In some embodiments, it involves adding DNA barcodes or tags to cDNA molecules as they are generated from mRNA. Nested PCR can be performed to minimize PCR amplification bias. Adapters can be added for sequencing, for example, using next-generation sequencing (NGS). Sequencing results can be used to determine the sequence of nucleotide fragments of one or more copies of the cellular label, molecular label, and target.

[0082] In certain embodiments, the provided method further comprises subjecting the prepared expression library, for example, the amplicon composition produced as described above, to a sequencing protocol, for example, an NGS protocol. The protocol can be performed on any suitable NGS sequencing platform. NGS sequencing platforms of interest include, but are not limited to, sequencing platforms provided by Illumina® (e.g., HiSeq™, MiSeq™, and / or NextSeq™ sequencing systems); Ion Torrent™ (e.g., Ion PGM™ and / or Ion Proton™ sequencing systems); Pacific Biosciences (e.g., PACBIO RS II sequencing systems); Life Technologies™ (e.g., SOLiD sequencing systems); Oxford Nanopore (e.g., Minion), Roche (e.g., 454 GS FLX+ and / or GS Junior sequencing systems); or any other sequencing platform of interest. The NGS protocol will vary depending on the specific NGS sequencing system used. Detailed protocols for sequencing, which may include, for example, further amplification (e.g., solid-phase amplification), sequencing of the amplicons, and analysis of the sequencing data, are available from the manufacturer of the NGS sequencing system used.

[0083] In some cases, the method further includes using an oligonucleotide-labeled cellular constituent-binding reagent, for example, in applications where detection, e.g., quantification, of one or more cellular constituents, e.g., surface proteins, is desired. The oligonucleotide-labeled cellular constituent-binding reagent used in such embodiments includes a cellular constituent-binding reagent, e.g., an antibody or binding fragment thereof, coupled to a cellular constituent-binding reagent-specific oligonucleotide that includes an identifier sequence for the cellular constituent-binding reagent with which the cellular constituent-binding reagent-specific oligonucleotide associates. In such cases, the magnetic capture beads may include nucleic acids configured to capture, e.g., specifically bind, a domain of the cellular constituent-binding reagent-specific oligonucleotide. In this way, protein expression may be assayed in conjunction with gene expression, for example, when multi-ohmic analysis, e.g., combined analysis of the transcriptome and proteome, is desired. In such cases, the method may include preparing the captured sample with the oligonucleotide-labeled cellular constituent-binding reagent, followed by capturing the cellular constituent-binding reagent-specific oligonucleotide released from the captured compartmentalized cells. Further details regarding the use of oligonucleotide-labeled cellular component binding reagents can be found in U.S. Patent Application Publication Nos. 20180267036 and 20200248263, the disclosures of which are incorporated herein by reference.

[0084] For example, as noted above, further details regarding methods for obtaining sequence data from single cells are provided in U.S. Patent Application Publication Nos. 2018 / 0088112; 2018 / 0200710; 2018 / 0346970; 2019 / 0056415; 2020 / 0248263; 2020 / 0299672; and 2021 / 0171940, the disclosures of which are incorporated herein by reference.

[0085] Representative Embodiments One embodiment of the method of the present invention is illustrated schematically in Figures 2A and 2B. As shown in Figure 2A, double emulsion single-cell droplets are first produced. The double emulsion single-cell droplets are water-in-oil-in-water double emulsion droplets. The droplets have a diameter of 38 μm, which corresponds to the diameter of the microwell into which the droplets are introduced. Following production of the initial composition of double emulsion single-cell droplets, the droplets of the initial composition are sorted using a standard FACS machine to produce a composition in which substantially all droplets contain a single cell. A FACS step is performed to remove empty droplets, thereby producing a composition enriched from droplets containing single cells. As shown in Figure 2B, the resulting composition enriched in droplets containing single cells is then loaded into a BD Rhapsody™ Express (Becton Dickinson and Company) instrument and operated according to the manufacturer's protocol (BD Rhapsody™ Single-Cell Analysis System Instrument User Guide, February 2019, Becton Dickinson and Company) to deposit double emulsion single-cell droplets into the microwells of the cartridge. As shown, substantially all of the microwells of the cartridge contain single cells containing double emulsion droplets. The droplets are then disrupted to release the cells by breaking the emulsion, for example, by contacting the emulsion with SDS. After cell release, barcoded beads are loaded into the microwells, thereby producing a compartmentalized single cell / barcoded bead composition. The above embodiment can provide better loading of cells / barcoded beads into compartments than Poisson loading and offers a substantial improvement over existing protocols.

[0086] kit Aspects of the present invention further include kits and compositions used to carry out various embodiments of the methods of the present invention. Kits of the present invention may include one or more components that find use in carrying out embodiments of the methods. For example, kits may include components used in producing double emulsion single-cell droplet compositions, such as an aqueous phase, an oil phase, a carrier phase, a microfluidic device, a surfactant, etc. Additionally, kits may include one or more components used to obtain sequence data, such as primers, polymerases (e.g., thermostable polymerases, reverse transcriptases (both with hot-start properties), etc.), dsDNAse, exonucleases, dNTPs, metal cofactors, one or more nuclease inhibitors (e.g., RNase inhibitors and / or DNase inhibitors), one or more molecular crowding agents (e.g., polyethylene glycol), one or more enzyme stabilizing components (e.g., DTT), a stimuli-responsive polymer, or any other desired kit component(s), such as one or more of the devices, solid supports, containers, cartridges, such as tubes, beads, plates, microfluidic chips, etc., as described above. The components of the kit may be present in separate containers, or multiple components may be present in a single container.

[0087] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for carrying out the subject methods. These instructions may be present in the subject kits in various forms, one or more of which may be present in the kit. One form in which these instructions may be present is information printed on a suitable medium or substrate, such as one or more pieces of paper on which the information is printed, kit packaging, package inserts, etc. Another form in which these instructions may be present is a computer-readable medium on which the information is recorded, such as a diskette, a compact disc (CD), a portable flash drive, etc. Another form in which these instructions may be present is a website address that can be used via the Internet to access the information at the removed site.

[0088] Notwithstanding the scope of the appended claims, the present disclosure is also defined by the following clauses.

[0089] 1. A method for producing a compartmentalized single cell / barcoded bead composition, comprising: contacting the encapsulated single cell composition with a plurality of microwells such that at least a portion of the plurality of microwells contain a single deposited encapsulated single cell; and introducing a barcoded bead into the microwell containing the deposited single cell to produce a compartmentalized single cell / barcoded bead composition.

[0090] 2. The method of clause 1, wherein the encapsulated single cell has a size corresponding to the size of the microwell such that only one encapsulated single cell can fit within the microwell.

[0091] 3. The method according to clause 2, wherein the size of the encapsulated single cells is in the range of 10 to 100 µm.

[0092] 4. The method of any one of the preceding clauses, wherein the contacting comprises flowing the encapsulated single-cell composition across the openings of the plurality of microwells.

[0093] 5. The method of clause 4, wherein the encapsulated single-cell composition is flowed across the openings of the plurality of microwells at a rate ranging from 1 μl / s to 5 ml / s.

[0094] 6. The method of any one of the preceding clauses, wherein the contacting causes a majority of the plurality of microwells to contain a single deposited encapsulated single cell.

[0095] 7. The method of clause 6, wherein the majority comprises 75% or more of the plurality of microwells.

[0096] 8. The method of clause 7, wherein the majority comprises 90% or more of the plurality of microwells.

[0097] 9. The method of clause 8, wherein the majority includes substantially all of the plurality of microwells.

[0098] 10. The method of any one of the preceding clauses, wherein the encapsulated single cell comprises a gel-encapsulated single cell.

[0099] 11. The method of any one of clauses 1-9, wherein the encapsulated single cell comprises a double emulsion single cell droplet.

[0100] 12. The method of clause 11, wherein the composition of the double emulsion single cell droplets comprises water-in-oil-in-water double emulsion single cell droplets.

[0101] 13. The method of any one of clauses 11 or 12, further comprising disrupting the deposited double emulsion single cell droplets to generate microwells containing released single cells.

[0102] 14. The method of clause 13, wherein the disrupting comprises contacting the deposited double emulsion single-cell droplets with a disrupting agent to release the cells of the droplets.

[0103] 15. The method of clause 14, wherein the disrupting agent comprises a chemical disrupting agent.

[0104] 16. The method of clause 14, wherein the disruptive agent comprises pressure.

[0105] 17. The method of any one of clauses 13-16, further comprising washing the released cells before introducing the barcoded beads.

[0106] 18. The method of any one of the preceding clauses, wherein the introducing comprises flowing a composition of barcoded beads across the opening of the microwell containing the deposited single cell.

[0107] 19. The method of clause 18, wherein the barcoded bead composition is flowed across the opening of the microwell containing the released single cells at a rate ranging from 1 μl / sec to 5 ml / sec.

[0108] 20. The method of any one of the preceding clauses, wherein the majority of the microwells of the plurality of microwells contain a single cell and a single bead by introducing.

[0109] 21. The method of clause 20, wherein the majority comprises 75% or more of the plurality of microwells.

[0110] 22. The method of clause 21, wherein the majority comprises 90% or more of the plurality of microwells.

[0111] 23. The method of clause 22, wherein the majority includes substantially all of the plurality of microwells.

[0112] 24. The method of any one of the preceding clauses, wherein the microwells of the plurality of microwells have diameters in the range of 5 μm to 100 μm.

[0113] 25. The method of any one of the preceding clauses, wherein the plurality of microwells comprises a microwell array.

[0114] 26. The method of clause 25, wherein the microwell array comprises between 100 and 12,500 microwells.

[0115] 27. The method of any one of the preceding clauses, wherein the plurality of microwells is disposed on the bottom surface of the flow cell.

[0116] 28. The method of any one of the preceding clauses, comprising evaluating a plurality of microwells after contacting.

[0117] 29. The method of any one of the preceding clauses, comprising evaluating a plurality of microwells after the introduction.

[0118] 30. The method of any one of clauses 28-29, wherein assessing comprises imaging.

[0119] 31. The method of any one of the preceding clauses, further comprising producing encapsulated single-cell droplets.

[0120] 32. The method of clause 31, wherein the producing step includes preparing an initial composition of encapsulated single cells and selecting encapsulated single cells of interest from the initial composition to produce the composition of encapsulated single cells.

[0121] 33. The method of clause 32, wherein selecting comprises cell sorting.

[0122] 34. The method of clause 33, wherein cell sorting comprises fluorescence-activated cell sorting (FACS).

[0123] 35. The method of any one of the preceding clauses, further comprising lysing the cells of the compartmentalized single cell / barcoded bead composition to release nucleic acids from the cells and produce barcoded beads comprising the hybridized released nucleic acids.

[0124] 36. The method of clause 35, further comprising preparing a sequenceable nucleic acid library from the barcoded beads containing the hybridized released nucleic acids.

[0125] 37. The method of clause 36, further comprising sequencing the nucleic acid library.

[0126] 38. The method of clause 37, wherein sequencing comprises next-generation sequencing (NGS).

[0127] 39. A microwell array, wherein a majority of the microwells of the microwell array contain both single cells and barcoded beads.

[0128] 40. The microwell array of clause 39, wherein the majority comprises 75% or more of the microwells of the array.

[0129] 41. The microwell array of clause 40, wherein the majority comprises 90% or more of the microwells of the array.

[0130] 42. The microwell array of clause 41, wherein the majority comprises substantially all of the microwells of the array.

[0131] 43. The microwell array of any one of clauses 39 to 42, which is present on the bottom surface of a flow cell.

[0132] Although the foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, it will be readily apparent to those skilled in the art that certain changes and modifications can be made in light of the teachings of the invention without departing from the spirit or scope of the appended claims.

[0133] Accordingly, the foregoing merely illustrates the principles of the present invention. It will be appreciated that those skilled in the art will be able to devise various configurations, not explicitly described or shown herein, which embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts the inventors have contributed to advancing the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended as a dedication to the public, regardless of whether such disclosure is expressly recited in the claims.

[0134] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) are expressly defined as being invoked for a limitation in a claim only if the exact phrase "means for" or the exact phrase "step for" appears at the beginning of such limitation in the claim. If such exact phrases are not used in a claim limitation, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is not invoked.

[0135] This application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 396,747, filed August 10, 2022, the disclosure of which is incorporated herein by reference.

Claims

1. 1. A method of making a compartmentalized single cell / barcoded bead composition, comprising: contacting the encapsulated single cell composition with a plurality of microwells such that at least some of the plurality of microwells contain a single deposited encapsulated single cell; and introducing a barcoded bead into the microwell containing the deposited single cell to produce a compartmentalized single cell / barcoded bead composition.

2. 10. The method of claim 1, wherein the encapsulated single cell has a size that corresponds to the size of the microwell such that only one encapsulated single cell can fit within the microwell.

3. 3. The method of claim 1 or 2, wherein said contacting comprises flowing said encapsulated single-cell composition across openings of said plurality of microwells.

4. 4. The method of claim 1, wherein said contacting causes a majority of said plurality of microwells to contain a single deposited encapsulated single cell.

5. 9. The method of claim 8, wherein said majority comprises substantially all of said plurality of microwells.

6. The method of any one of claims 1 to 5, wherein the encapsulated single cell comprises a gel-encapsulated single cell.

7. 6. The method of any one of claims 1 to 5, wherein the encapsulated single cells comprise double emulsion single-cell droplets.

8. 8. The method of claim 7, wherein the composition of the double emulsion single cell droplets comprises water-in-oil-in-water double emulsion single cell droplets.

9. 9. The method of claim 7 or 8, further comprising disrupting the deposited double emulsion single-cell droplets to generate microwells containing released single cells.

10. 10. The method of claim 9, wherein said disrupting comprises contacting the deposited double emulsion single-cell droplets with a disrupting agent to release cells of the droplets.

11. The method of any one of claims 7 to 10, further comprising washing the released cells before introducing the barcoded beads.

12. 12. The method of any one of claims 1-11, wherein said introducing comprises flowing a composition of barcoded beads across the opening of a microwell containing the deposited single cell.

13. 13. The method of any one of claims 1 to 12, wherein said introducing causes a majority of said microwells of said plurality of microwells to contain a single cell and a single bead.

14. 14. The method of any one of claims 1 to 13, wherein the plurality of microwells comprises a microwell array.

15. 1. A microwell array comprising: A microwell array, wherein a majority of the microwells of said microwell array contain both single cells and barcoded beads.

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