Core-shell gel particles for separating and analyzing RNA and DNA from a cell

Abstract

Provided herein are compositions, systems, kits, and methods for separating and analyzing RNA / cDNA from genomic DNA molecules using core-shell gel particles (e.g., such that separate barcoded sequencing libraries can be generated from each). In certain embodiments, the core-shell gel particles comprise an outer gel shell, and an interior region surrounded by said shell that comprises: A) an interior solution that is at least partially aqueous, B) optionally an inner gel core; C) at least one cell, and D) a plurality of capture beads comprising capture probes configured to hybridize to RNA from the cell, and optionally configured to hybridize to genomic DNA and / or bind target proteins.

Description

[0001] CORE-SHELL GEL PARTICLES FOR SEPARATING AND ANALYZING RNA and DNA FROM A CELL

[0002] The present application claims priority to U.S. Provisional application serial number 63 / 491,215, filed March 20, 2023, which is herein incorporated by reference in its entirety.

[0003] FIELD OF THE INVENTION

[0004] Provided herein are compositions, systems, kits, and methods for separating and analyzing RNA / cDNA from genomic DNA molecules using core-shell gel particles (e.g., such that separate barcoded sequencing libraries can be generated from each). In certain embodiments, the core-shell gel particles comprise an outer gel shell, and an interior region surrounded by said shell that comprises: A) an interior solution that is at least partially aqueous, B) optionally an inner gel core; C) at least one cell, and D) a plurality of capture beads comprising capture probes configured to hybridize to RNA (e.g., mRNA) from the cell, and optionally configured to hybridize to genomic DNA and / or bind target proteins.

[0005] BACKGROUND

[0006] The field of single cell analysis had enabled researchers to understand the diversity in phenotype and genotype among populations of cells. Flow cytometry has enabled individual cells to be profiled based on surface markers. Recent advances in microfluidics have enabled RNA, DNA, and epigenetic sequencing on a single cell level, leading to commercially available products and widespread adoption. Microfluidics have also enabled single cells to be isolated and assayed for >20 secreted proteins. Further advances in the field have led to the field of single cell multi-modal omics, the measurement of multiple different types of data from the same single cells. What is needed are methods that allow RNA and DNA from a single cell to be separately analyzed, such as by sequencing, without laborious and complex multi-step processes.

[0007] SUMMARY OF THE INVENTION

[0008] Provided herein are compositions, systems, kits, and methods for separating RNA / cDNA from genomic DNA molecules using core-shell gel particles (e.g., such that separate barcoded sequencing libraries can be generated from each). In certain embodiments, the core-shell gel particles comprise an outer gel shell, and an interior region surrounded by said shell that comprises: A) an interior solution that is at least partially aqueous, B) optionally an inner gel core; C) at least one cell, and D) a plurality of capture beads comprising capture probes configured to hybridize to RNA (e.g., mRNA) from the cell, and optionally configured to hybridize to genomic DNA and / or bind target proteins.

[0009] In some embodiments, provided herein are compositions, systems, and kits comprising: a) a plurality of core-shell gel particles that each comprise: i) an outer gel shell, and ii) an interior region, surrounded by the shell, that comprises: A) an interior solution that is at least partially aqueous, B) optionally an inner gel core; C) the at least one cell, and D) a plurality of capture beads comprising: I) capture probes configured to hybridize to RNA (e.g., mRNA) from the cell, and optionally II) DNA capture sequences configured to hybridize to DNA from the cell; and optionally III) protein binding agents; and b) optionally a lysing agent solution. In particular embodiments, the compositions, system, and kits further comprise an aqueous solution or an oil carrier phase, wherein the plurality of core- shell gel particles are present in the aqueous solution or the oil carrier phase.

[0010] In certain embodiments, provided herein methods comprising: a) generating an emulsion comprising a plurality of aqueous droplets in an oil phase such that at least some of the aqueous droplets are positive droplets that each comprise: i) an aqueous solution; ii) a gelling agent (e.g., hydrogel, chitosan, etc.), iii) at least one cell, and iv) optionally a plurality of capture beads comprising: A) capture probes configured to hybridize to RNA (e.g., mRNA) from the cell, and optionally B) DNA capture sequences configured to hybridize to DNA from the cell; and optionally C) protein binding agents; b) treating the positive droplets (e.g., optionally by cooling) in the emulsion such that the gelling agent gels (e.g., by UV light or by cooling) in a plurality of the positive droplets thereby forming a plurality of core-shell gel particles that are present in the oil phase and that each comprise: i) an outer shell, and ii) an interior region, surrounded by the shell, that comprises: A) an interior solution that is at least partially aqueous, B) optionally an inner gel core; C) the at least one cell, and D) optionally at least some of the capture beads, and c) adding a lysing agent solution to the emulsion such that at least part of the lysing solution agent migrates through the outer shell into the interior region and lyses the at least one cell releasing RNA and / or DNA molecules in each of the plurality of core-shell gel particles. In some embodiments, the gelling agent (e.g., chitosan) used to form the outer gel shell and / or inner gel core comprises additives such as chitosan or other moictics. These additives can serve various purposes such as, for example, enhancing nucleic acid capture, improving gel strength or porosity, or providing other desirable chemical or physical properties to the gel particles.

[0011] In some embodiments, the plurality of capture beads are present but without the DNA capture sequences, and the method further comprises: d) incubating the emulsion such that in each of the plurality of core-shell gel particles: A) at least some of the RNA molecules specifically hybridize to the capture probes thereby generating RNA-bound capture beads, and B) the DNA molecules are present in the interior solution and do not specifically hybridize to the capture probes, and do not migrate out of the outer shell; and e) optionally adding reversetranscription reagents to the emulsion to generate cDNA from the RNA bound to the RNA- bound capture beads. In further embodiments, the methods further comprise: e) transferring at least some of the plurality of core-shell gel particles from the oil phase into an aqueous carrier, and f) treating the plurality of core-shell gel particles to generate a first sample and a second sample, wherein the first sample comprises the RNA-bound capture beads and is free or substantially free of the DNA molecules, and wherein the second sample comprises all or substantially all of the DNA molecules and is free or substantially free of the RNA molecules.

[0012] In other embodiments, the capture probes are attached to the capture beads via a reversible linker, and the method further comprises: g) treating the first sample such that the RNA molecules are reverse transcribed into cDNA either on the capture beads or after having been cleaved from the capture beads via the reversible linker. In additional embodiments, the methods further comprise: h) generating a sequencing library from the cDNA by attaching adapters to the cDNA or amplicons thereof and amplifying the cDNA and / or the amplicons using primers, wherein optionally the capture probes and / or the adapter comprise a barcode sequence, and i) sequencing the sequencing library to generate a plurality of RNA / cDNA sequencing reads. In additional embodiments, the methods further comprise: h) generating a sequencing library from the DNA molecules in the second sample, by attaching adapters to the DNA or amplicons thereof and amplifying the DNA molecules and / or the amplicons using primers, wherein optionally the adapters comprise a barcode sequence, and i) sequencing the sequencing library to generate a plurality of DNA sequencing reads. In some embodiments, the plurality of capture beads are present with the DNA capture sequences, and the method further comprises: d) incubating the emulsion such that in each of the plurality of core-shell gel particles: A) at least some of the RNA molecules specifically hybridize to the capture probes, and B) at least some of the DNA molecules specifically or non- specifically hybridize to the DNA capture sequences; thereby generating RNA + DNA bound capture beads. In additional embodiments, the methods further comprise: e) transferring at least some of the plurality of core-shell gel particles from the oil phase into an aqueous carrier, and f) treating the plurality of core-shell gel particles to generate a first sample comprising purified or substantially purified RNA + DNA bound capture beads. In additional embodiments, the capture probes are attached to the capture beads via a reversible linker, and further comprising: g) at least one of the following: A) cleaving the RNA molecules from the RNA + DNA capture beads via the reversible linker to generate free RNA and DNA-capture beads, and separating the free RNA into a second sample and the DNA-capture beads into a third sample, and reverse transcribing the free RNA in the second sample into cDNA; or B) reverse transcribing the RNA on the RNA + DNA capture molecules into cDNA molecules, and cleaving the RNA and cDNA molecules from the RNA + DNA capture beads via the reversible linker to generate free RNA, free cDNA, and DNA-capture beads, and separating the free RNA and free cDNA into a second sample and the DNA-capture beads into a third sample.

[0013] In particular embodiments, the methods further comprise: h) generating a first sequencing library from the cDNA from the second sample by attaching adapters to the cDNA or to amplicons thereof, and amplifying the cDNA and / or amplicons thereof using primers, wherein optionally the capture probes and / or the adapters each comprise a barcode sequence, and i) sequencing the first sequencing library to generate a plurality of RNA / cDNA sequencing reads. In additional embodiments, the methods further comprise: h) generating a second sequencing library from the DNA molecules in the third sample by attaching adapters to the DNA molecules or to amplicons thereof, and amplifying the DNA molecules and / or amplicons using primers, wherein optionally the capture sequences and / or the adapters each comprise a barcode sequence and i) sequencing the second sequencing library to generate a plurality of DNA sequencing reads.

[0014] In some embodiments, the plurality of capture beads are not present, and the method further comprises: d) incubating the emulsion such that substantially all of the RNA molecules in each of the plurality of core-shell particles migrate out of the interior region through the outer shell and such that substantially all of the DNA molecules remain inside the outer shell in each of the core-shell particles. In certain embodiments, the methods further comprise: e) transferring at least some of the plurality of core-shell gel particles from the oil phase into an aqueous carrier, and f) treating the plurality of core-shell gel particles to generate a first sample comprising purified or substantially purified DNA molecules. In additional embodiments, the methods further comprise: f) generating a sequencing library from the DNA molecules in the first sample by attaching adapters to the DNA molecules or to amplicons thereof, and amplifying the DNA molecules and / or amplicons using primers, wherein optionally the adapters each comprise a barcode sequence and i) sequencing the sequencing library to generate a plurality of DNA sequencing reads.

[0015] In certain embodiments, the at least one cell in each of the plurality of core- shell gel particles is one and only one cell. In additional embodiments, the generating the emulsion comprises combining an aqueous phase with the oil phase in bulk solution, wherein the aqueous phase comprises: i) an aqueous solution; ii) the gelling agent (e.g., chitosan) iii) a plurality of cells, and iv) optionally a plurality of the capture beads.

[0016] In some embodiments, the generating the emulsion comprises: i) forming a precursor emulsion comprising a plurality of precursor aqueous droplets in an oil phase that each contain a precursor core-shell gel particle, ii) combining said plurality of precursor aqueous droplets with a solution comprising cells to generate a precursor solution, iii) heating said precursor solution such that said precursor core-shell gel particle in each of said precursor aqueous droplets melts forming gelling agent (e.g., chitosan) and mixes with at least one cell, thereby forming said plurality of aqueous droplets recited in step a) above. In other embodiments, generating the emulsion comprises: combining a first aqueous phase with the oil phase using a microfluidic droplet maker, wherein: I) the first aqueous phase comprises one or more of the following: i) an aqueous solution; ii) the gelling agent, iii) a plurality of cells, and iv) optionally a plurality of the capture beads, or II) wherein a second aqueous phase is combined with the first aqueous phase and the oil phase to generate the emulsion, and wherein the second aqueous phase comprises one or more of the following: i) an aqueous solution; ii) the gelling agent, iii) a plurality of cells, and iv) optionally a plurality of the capture beads. In other embodiments, the microfluidic droplet maker comprises a co-flow droplet maker. In additional embodiments, the microfluidic droplet maker comprises: a flow focusing component, a T-junction, a step emulsification component, or a jet triggered droplet generator. In further embodiments, the microfluidic droplet maker employs particle-templated emulsification or geometrically mediated breakup in a hierarchical array and optionally without requiring the substantial control of flow rate.

[0017] In certain embodiments, the positive droplets further comprise cell media. In additional embodiments, the positive droplets comprise at least two cells, or only two cells, or only three cells. In further embodiments, the positive droplets have a diameter of from about 1 pm to 1000 pm. In additional embodiments, the lysing agent solution comprises a lysing agent, wherein the lysing agent is optionally a protease. In other embodiments, the lysing agent solution comprises a detergent or other chemical that overcomes inhibition and optionally facilitate barcoding of nucleic acids and / or micelle transport. In some embodiments, the amplifying is performed by a temperature-cycling methodology (e.g., PCR) or an isothermal methodology (e.g., multiple displacement amplification (MDA)), and / or is performed before or after the attachment of adapters. In other embodiments, the sequencing is performed by a method selected from: Ab-seq, CITE-seq, sequencing by synthesis, and next generation sequencing.

[0018] In particular, embodiments, the plurality of capture beads are present and comprise the protein binding agents. In other embodiments, the protein binding agents bind target proteins from the at least one cell, and optionally wherein the target proteins are purified into a purified protein sample. In other embodiments, the emulsion is a single emulsion or double emulsion. In additional embodiments, the methods further comprise: sorting the plurality of core-shell gel particles based on the presence or absence of a signal or detectable molecule from each of the plurality of core-shell gel particles. In further embodiments, the sorting is based on the presence or absence of the detectable molecule are particular barcoded or unbarcoded DNA or RNA molecules.

[0019] DEFINITIONS

[0020] As used herein, a bead is the platform upon which nucleic acid and / or protein capture probes and / or primers are attached or linked to. A bead may be any desired shape. The term “beads” include pellets, disks, fibers, gels, particles pads, slides, matrices, etc. A bead state may be solid, porous, rigid, semi-rigid, deformable, hard, etc. Bead material may be any desired composition that maintains the mechanical integrity of a bead, is compatible with the nucleic acid primers and capture sequences or protein capture molecules. A bead surface composition may include, for example, cellulose, glass, porcglass, resin, silica, polystyrene, or other suitable materials.

[0021] DETAILED DESCRIPTION

[0022] Provided herein are compositions, systems, kits, and methods for separating and analyzing RNA / cDNA from genomic DNA molecules using core-shell gel particles (e.g., such that separate barcoded sequencing libraries can be generated from each). The technology solves the problem of analyzing both RNA and DNA from cells without the need for multi-step, complex processing methodologies. In certain embodiments, the core-shell gel particles comprise an outer gel shell, and an interior region surrounded by said shell that comprises: A) an interior solution that is at least partially aqueous, B) optionally an inner gel core; C) at least one cell, and D) a plurality of capture beads comprising capture probes configured to hybridize to m RNA (e.g., mRNA) from the cell, and optionally configured to hybridize to genomic DNA and / or bind target proteins. Droplets may be generated in solution (e.g., suspension) or in microfluidic devices.

[0023] In certain embodiments, a lysing agent is added into the carrier phase and transported into the droplets by micellular transport, causing the cells to lyse and release their RNA (e.g., mRNA) which is captured on the barcode bead. The resultant emulsions can then be solidified to harden the shell and thereby sterically capture the genomic DNA in the shell and around the bead. These core- shell structures can then be transferred out of the oil and into a new aqueous phase where they may be subjected to cDNA synthesis by transfer into reverse transcription reagent and incubating to allow the reverse transcription reaction to occur. Washes can be performed to remove background material and the now barcoded cDNA can be released by using a reversible linker on the hydrogel bead such as a USER-digestible linker, or a photodegradable linker and applying light. The cDNA products are allowed to diffuse out of the hydrogels core-shell particles and can be recovered in the supernatant for sequencing library preparation and sequencing.

[0024] In particular embodiments, the core-shell particles containing the RNA-barcoded beads are mixed with a PCR solution that also contains the primers necessary for the multiplexed DNA amplification of the desired targets in the genomic DNA. These primers can anneal with barcodes on the hydrogels to produce PCR products that are barcoded with the same barcode that was transferred to the cDNA during the RNA barcoding step. In particular embodiments, to perform the genomic DNA barcoding, the particles are re-encapsulated into oil by solvent transfer, such as emulsification, and then thermal cycled to perform the DNA amplification of the target regions of interest. This yields the desired barcoded PCR products. Finally, the emulsion is broken and the PCR products recovered and then used for sequence library preparation and sequencing. Thus, this approach barcodes RNA using a barcode bead in one step, and barcodes DNA using the same barcode bead in a separate step. By utilizing the ability to release primers from the beads at specific times, such as after cDNA synthesis and after adding PCR primers, this process allows for simultaneous RNA and DNA sequencing at the single cell level.

[0025] In particular embodiments, one factor in this process is the removal of unbound cDNA that may have been generated prior to the execution of the genomic DNA PCR. This is because cDNA may have homology to sequences targeted in the genomic DNA, and therefore may interfere with those reactions, reducing the quality of the resulting DNA data. To ensure that this does not happen, it is generally important to remove the cDNA prior to genomic DNA amplification. This can be accomplished with rigorous washes to remove the unbound cDNA. Alternatively, the primers used to generate the cDNA products can be selected so as to allow denaturation with an enzyme, thus ensuring that any cDNA not recovered during the washing step is removed prior to the DNA amplification step.

[0026] In some embodiments, the methods herein allow for capturing RNA (e.g., mRNA) that can be combined with DNA and protein sequencing approaches as desired to perform multi- omics. Multiple approaches may be employed to allow capture of mRNA with this method. For example, in one embodiment, a process is described in which mRNA hybridizes to the bead and is reverse transcribed onto the bead to generate the barcoded cDNA products (“drop seq”). Alternatively, another approach is to release Poly T capture probes upon lysis of the cell such that the Poly T capture probes can bind to the mRNA of the lysed cell in solution, rather than on the surface of the bead (InDrops). In certain embodiments, if the reverse transcriptase enzyme and necessary reagents are present, this results in cDNA synthesis of the mRNA while it is tethered to the bead, thereby barcoding the mRNA. The resultant water-in-oil emulsions can then be broken and transferred into an aqueous phase, upon which the barcoded mRNA-cDNA products will diffuse out of the particles and can be recovered for sequencing. Provided the gel shell is present, the genomic DNA of the cell will remain with the barcode bead and can thus be processed for DNA sequencing. This is an approach for processing mRNA that includes performing the reverse transcription step with the lysis step to allow it to occur in solution rather than on the surface of the bead.

[0027] This certain embodiments, the methods herein involve first amplifying the DNA to generate barcoded products, and then creating cDNA products from the thermal cycled mRNA. This is accomplished, for example, by performing all the requisite steps for cell lysis, including attaching the resulting mRNA to beads through a process of hybridization with barcoded probes. At this stage, cDNA synthesis is not performed so as to not produce products that may interfere with the genomic DNA amplification. Next, the core-shell hydrogels are processed as necessary to perform targeted DNA amplification and barcoding, which involves the use of primers specific to different targets that have been barcoded to generate barcoded amplicons. During this process, mRNA and protein tags may be present in the solution, and will also be thermal cycled. However, since they are not DNA targets that are compatible with the PCR primers, they do not interfere with the reaction. After the DNA targets are recovered, the mRNA binds to the capture probes on the beads allowing for the beads to be washed to recover the DNA amplicons, and then processed for cDNA synthesis. The DNA targets can be separately prepared for sequencing and sequenced, while the mRNA, now bound to the beads, can be reverse transcribed into cDNA and barcoded for sequencing in a separate library preparation step. A potential challenge in this approach is that the mRNA is present during the thermal cycling for the DNA amplification, which may result in fragmentation of the mRNA and yield shorter product. However, methods to suppress RNA fragmentation, such as adjusting concentrations of ions in the PCR buffer and reducing annealing temperature can be utilized to help maintain the integrity of the mRNA and make it suitable for sequencing. Indeed, thermal fragmentation is a standard approach for mRNA sequencing.

[0028] In some embodiments, the aqueous droplets herein are flowed in a microfluidic device, which may be used to combine multiple drops such that all the reagents are combined into a single aqueous droplet. In certain embodiments, the microfluidic devices employed herein comprise a microenvironment on Demand (MOD) device, described in PCT application W02020232072A1 and Cole et al., Proc. Natl. Acad. Sci., 114(33): 8728-8733, 2017, which are both incorporated by reference herein in their entireties. In certain embodiments, the MOD platform is composed of a combination of deterministic single-cell droplet sorter and dropletassembler that can selectively assemble cells and reagents. MOD performs a cyclic buildup and release of designer droplets through the merging of select droplets on a defined dielectrophoretic trapping position inside the microfluidic device. This approach is advantageous because it is less prone to contamination, higher throughput, and requires fewer moving parts than other devices. The flexible nature of the MOD platform makes it a well- suited technology to perform integrated and functional cell-cell, cell-ECM interaction analysis and link any perturbations to select expressed gene sequences or transcriptome profiles at a single cell level. Essentially, MOD allows for precise, flexible, scalable liquid handling that can build a large number of predetermined reaction conditions. MOD not only allows for the sorting and combination of particulates (e.g., cells, capture beads, liquid gelling precursor, etc.), but also sorts and assembles diverse droplet contents. Furthermore, droplets constructed with MOD are compartmentalized and miniaturized (e.g., -100 pL) providing contained reactions in concentrated volumes. These two aspects of MOD, reagent selection and reaction miniaturization, provide a powerful approach to phenotypically screen large numbers of single cells.

[0029] In certain embodiments, the MOD platform is employed to generate the aqueous droplets herein and droplet manipulation and sorting is achieved by electrowetting, the modification of the wetting properties of a surface with an applied electric field. Electrowetting manipulation of droplets in a microfluidic device may be achieved through the application of differential voltages to different regions in an electrode grid (see, US Pat. 6,911,132, herein incorporated by reference). Alternatively, droplet actuation and sorting can be achieved using optoelectrowetting, where localized electric fields are triggered through the selective application of light to a photoconductive layer (see, US Pat. 6,958,132, which is herein incorporated by reference in its entirety).

[0030] In certain embodiments, droplet-based cell culture or RNA and DNA sequencing library prep is performed using porous materials. The duration of cell culture in sub-nanoliter droplets is limited by a finite amount of encapsulated media and localized buildup of metabolic waste products. In cases where longer duration incubations are desired or required, it may be appropriate to convert a droplet to a media-permeable format while keeping encapsulated objects in place. This can be achieved by flowing hydrogel (liquid gel) precursors into droplets along with cells, then triggering gelation to form either gel beads or permeable capsules. After gelation, in certain embodiments, the emulsion is broken, the emulsion oil is removed, and the cell-laden capture-shells are suspended in media and cultured for a time. Examples of the hydrogel bead approach are given in Wan et al., (Polymers (Basel)., vol. 4, no. 2, pp. 1084-1108, 2012), Utech et al., (Adv. Healthc. Mater., 2015), and Dolega et al. (Biomaterials, vol. 52, no. 1, pp. 347-357, 2015.), all of which are herein incorporated by reference in their entireties. Examples of permeable capsules are given by Yu et al, (Biomed. Microdevices, vol. 17, no. 2, 2015.), van Loo et al (Mater. Today Bio, vol. 6, no. February, p. 100047, 2020.), and Leonaviciene et al. (Lab Chip, no. Advanced Article, 2020), all of which are herein incorporated by reference in their entireties. Extended cell culture (e.g., after a target protein detection assay as described herein) is especially useful in cases where cell proliferation is important, such as clonal expansion of single cells and cell-cell interaction assays where proliferation is a readout. In some cases, it may be desired to break down a gel bead or capsule via chemical, enzymatic, or thermal means in order to access the contents for further processing.

[0031] Aqueous droplets as used or generated in connection with the subject methods, devices, and / or systems may be sphere shaped or they may have any other suitable shape, e.g., an ovular or oblong shape. Aqueous droplets as described herein may include a liquid phase and / or a solid phase material. In some embodiments, aqueous droplets according to the present disclosure include a gel material. In certain embodiments, the aqueous droplets comprise double emulsions (or multiple emulsion) or are treated to generate hydrogel shells.

[0032] Exemplary double and multiple emulsions are described in U.S. Pat. 9,238,206, which is herein incorporated by reference in its entirety, particularly for such double and multiple emulsions. In general a multiple emulsion describes larger droplets that contain one or more smaller droplets therein. In a double emulsion, the larger droplets may, in turn, be contained within another fluid, which may be the same or different than the fluid within the smaller droplet. In certain embodiments, larger degrees of nesting within the multiple emulsion are possible. For example, an emulsion may contain droplets containing smaller droplets therein, where at least some of the smaller droplets contain even smaller droplets therein, etc. In certain embodiments, a double emulsion is produced, e.g., a carrying fluid, containing a second fluidic droplet, which in turn contains a first fluidic droplet therein. In some cases, the carrying fluid and the first fluid may be the same. The fluids may be of varying miscibilities, e.g., due to differences in hydrophobicity. For example, the first fluid may be water soluble, the second fluid oil soluble, and the carrying fluid water soluble. This arrangement is often referred to as a w / o / w multiple emulsion (“water / oil / water”). Another double emulsion may include a first fluid that is oil soluble, a second fluid that is water soluble, and a carrying fluid that is oil soluble. This type of double emulsion is often referred to as an o / w / o double emulsion (“oil / water / oil”). It should be noted that the term “oil” in the above terminology merely refers to a fluid that is generally more hydrophobic and not miscible in water, as is known in the art. Thus, the oil may be a hydrocarbon in some embodiments, but in other embodiments, the oil may comprise other hydrophobic fluids.

[0033] In certain embodiments, the aqueous droplets herein comprise a hydrogel shell or microcapsule, such as exemplified in U.S. Pat. 10,710,045 and U.S. Pat. Pub. 20140127290, both of which are herein incorporated by reference in their entireties, particularly for such hydrogel shells or microcapsules. In certain embodiments, the hydrogel shells for the aqueous droplets, or microcapsules, comprise a liquid core, and at least one external envelope totally encapsulating the liquid core at its periphery, said external envelope being able to retain the liquid core when the capsule is immersed into a gas and comprising at least one gelled polyelectrolyte and / or a stiffened biopolymer. In certain embodiments, such microcapsules contain a cell and / or other reagents discussed herein. In certain embodiments, a microcapsule refers to a particle or capsule having a mean diameter of about 50 pm to about 1000 pm, formed of a cross-linked hydrogel shell surrounding a biocompatible matrix. The microcapsule may have any shape suitable for cell encapsulation. The microcapsule may contain one or more cells dispersed in the biocompatible matrix, cross-linked hydrogel, or combination thereof, thereby “encapsulating” the cells.

[0034] In some embodiments, the subject aqueous droplets have a dimension, e.g., a diameter, of or about 1.0 pm to 1000 pm, inclusive, such as 1.0 pm to 750 pm, 1.0 pm to 500 pm, 1.0 pm to 100 pm, 1.0 pm to 10 pm, or 1.0 pm to 5 pm, inclusive. In some embodiments, aqueous droplets as described herein have a dimension, e.g., diameter, of or about 1.0 pm to 5 pm, 5 pm to 10 pm, 10 pm to 100 pm, 100 pm to 500 pm, 500 pm to 750 pm, or 750 pm to 1000 pm, inclusive. Furthermore, in some embodiments, aqueous droplets as described herein have a volume ranging from about 1 fL to 1 nL, inclusive, such as from 1 fL to 100 pL, 1 fL to 10 pL, 1 fL to 1 pL, 1 fL to 100 fL, or 1 fL to 10 fL, inclusive. In some embodiments, aqueous droplets as described herein have a volume of 1 fL to 10 fL, 10 fL to 100 fL, 100 fL to 1 pL, 1 pL to 10 pL, 10 pL to 100 pL or 100 pL to 1 nL, inclusive. In addition, aqueous droplets as described herein may have a size and / or shape such that they may be produced in, on, or by a microfluidic device and / or flowed from or applied by a microfluidic device.

[0035] In some embodiments, the aqueous droplets as described herein refer to small, generally spherically structures, containing at least a first fluid phase, such as an aqueous phase (e.g., water), bounded by a second fluid phase (e.g., oil) which is immiscible with the first fluid phase. In some embodiments, droplets according to the present disclosure may contain a first fluid phase (e.g., oil) bounded by a second immiscible fluid phase (e.g., an aqueous phase fluid, such as water). In some embodiments, the second fluid phase is an immiscible phase carrier fluid. Thus, droplets according to the present disclosure may be provided as aqueous-in-oil emulsions or oil in aqueous emulsions. Droplets may be sized and / or shaped as described herein for aqueous droplets. For example, droplets according to the present disclosure generally range from 1 pm to 1000 pm, inclusive, in diameter. Droplets according to the present disclosure may be used to encapsulate cells, nucleic acids (e.g., DNA and / or RNA), enzymes, reporter dyes, reagents, and a variety of other components. The term droplet may be used to refer to a droplet produced in, on, or by a microfluidic device and / or flowed from or applied by a microfluidic device.

[0036] Aspects of the disclosed methods may include making aqueous droplets using one or more cells from a biological sample. In such cases, each aqueous droplet may contain zero, one, or more than one cell. In some cases, such aqueous droplets can be made by incorporating the biological sample, cells from the biological sample, lysate from cells of the biological sample, or any other sample derived from the biological sample into a mixed emulsion. In some cases, the method further includes separating one or more components of the biological sample or otherwise processing the biological sample (e.g. via centrifugation, filtration, and the like), before making the aqueous droplets.

[0037] Fluorescent tags (on the oligo probe, and / or dying a cell generally) can be used to image an aqueous droplet or combined aqueous droplet in the aqueous droplet merger region. Fluorescent tags can also be used to identify the particular type of aqueous droplets that were combined to create a given combined aqueous droplet. As such, the properties of the combined aqueous droplet or component thereof can be correlated with the contents that were used to make the original aqueous droplets. As an example, different types of cells can be labeled with different fluorescent tags and incorporated into aqueous droplets. After such cell-containing aqueous droplets arc combined with other aqueous droplets (c.g. containing other cells), the outcome of the combined aqueous droplets can be observed. As some of all of the original aqueous droplets can be labeled with fluorescent tags, the resulting combined aqueous droplet can have multiple fluorescent tags. In other cases, the combined aqueous droplet only has one fluorescent tag. Oligonucleotide barcodes can be used in a similar manner to that of fluorescent tags. Instead of detecting optical fluorescence, however, the oligonucleotide barcodes can be sequenced in order to identify the original aqueous droplets that formed the combined aqueous droplet.

[0038] Methods and devices which may be utilized in the encapsulating of a component from a biological sample are described in PCT Publication No. WO 2014 / 028378, the disclosure of which is incorporated by reference herein in its entirety and for all purposes. Encapsulation approaches of interest also include, but are not limited to, hydrodynamically-triggered drop formation and those described by Link, et al., Phys. Rev. Lett. 92, 054503 (2004), the disclosure of which is incorporated herein by reference. Other methods of encapsulating cells into droplets may also be applied. Where desired, the cells may be stained with one or more antibodies and / or probes prior to encapsulating them into drops.

[0039] One or more lysing agents may also be added to the aqueous droplets (e.g., droplets), containing a cell, under conditions in which the cell(s) may be caused to burst, thereby releasing their genomes and target proteins. The lysing agents may be in buffer containing detergents such as Triton XI 00 and / or proteinase K.

[0040] One or more primers may be introduced into the aqueous droplets or core- shell particles for each of the genes to be detected. Hence, in certain aspects, primers for all target genes (e.g., antibody genes) may be present in the aqueous droplet at the same time, thereby providing a multiplexed assay. The aqueous droplets or core- shell particles may be temperature-cycled so that they will undergo PCR. In certain embodiments, rolling circle amplification (RCA)-based proximity ligation is employed.

[0041] In some embodiments, a surfactant may be used to stabilize the aqueous droplets. In some cases, the aqueous droplets or the associated emulsion lack a surfactant. Accordingly, an aqueous droplet may involve a surfactant stabilized emulsion. Any convenient surfactant that allows for the desired reactions to be performed in the aqueous droplets, may be used. In other aspects, an aqueous droplet is not stabilized by surfactants or particles. The surfactant used depends on a number of factors such as the oil and aqueous phases (or other suitable immiscible phases (e.g., any suitable hydrophobic and hydrophilic phases)) used for the emulsions. For example, when using aqueous droplets in a fluorocarbon oil, the surfactant may have a hydrophilic block (PEG-PPO) and a hydrophobic fluorinated block (Krytox® FSH). If, however, the oil was switched to be a hydrocarbon oil, for example, the surfactant would instead be chosen so that it had a hydrophobic hydrocarbon block, like the surfactant AB IL EM90. In selecting a surfactant, desirable properties that may be considered in choosing the surfactant may include one or more of the following: (1) the surfactant has low viscosity; (2) the surfactant is immiscible with the polymer used to construct the device, and thus it doesn’t swell the device; (3) biocompatibility; (4) the assay reagents are not soluble in the surfactant; (5) the surfactant exhibits favorable gas solubility, in that it allows gases to come in and out; (6) the surfactant has a boiling point higher than the temperature used for PCR (e.g., 95°C); (7) the emulsion stability; (8) that the surfactant stabilizes drops of the desired size; (9) that the surfactant is soluble in the carrier phase and not in the droplet phase; (10) that the surfactant has limited fluorescence properties; and (11) that the surfactant remains soluble in the carrier phase over a range of temperatures.

[0042] The aqueous droplets (e.g., microdroplets) described herein may be prepared as emulsions, such as an aqueous phase fluid dispersed in an immiscible phase carrier fluid (e.g., a fluorocarbon oil or a hydrocarbon oil) or vice versa. In some cases, the carrier fluid comprises a fluorinated compound. In some cases, the carrier fluid is an aqueous fluid. The nature of the microfluidic channel (or a coating thereon) (e.g., hydrophilic or hydrophobic), may be selected so as to be compatible with the type of emulsion being utilized at a particular point in a microfluidic workflow.

[0043] In some cases, an aqueous droplet includes a capture bead. In some cases, at least one dimension of the capture bead (e.g., diameter, is between about 0.5 pm and about 500 pm). In some cases, the bead is made of a polymeric material, such as polystyrene. In some cases, the bead is magnetic or contains a magnetic component. In some cases, the bead has a biomolecule attached to its surface, such as poly-TTT oligos, an antibody, a protein, an antigen, DNA, RNA, streptavidin, or a combination thereof. As such, the present disclosure provides methods of selectively combining a biomolecule with another compound or cell, wherein the method includes selectively isolating the biomolecule (e.g., mRNA sequences from a lysed cell) from a composition using the capture bead, making an aqueous droplet that includes the bead and biomolecule, and selectively combining the aqueous droplet containing the bead and biomolecule with one or more other aqueous droplets that contain one or more other compounds or cells using microfluidic devices. Methods of selectively isolating biomolecules using capture beads are known in the ail, e.g. U.S. 2010 / 0009383, which is incorporated herein by reference for its disclosure of a method of separating a biomolecule or cell using beads.

[0044] In some embodiments, the methods, devices, and / or systems described herein can be used to sequence nucleic acid (e.g., separated mRNA / cDNA and genomic DNA molecules) derived from single cells (e.g., once they have been determined to secrete the target protein, such as a monoclonal antibody). For example, individual cells can be encapsulated in the core-shell particles which include the assay reagents as described herein. The cells can then be lysed and subjected to molecular biological processing to amplify and / or tag their nucleic acids with barcodes. Material that contains RNA / cDNA may be separated from genomic DNA molecules as described above into separate samples. The material from all the droplets can then be pooled for all cells and sequenced and the barcodes used to sort the sequences according to single droplets or cells. These methods can be used, for example, to sequence the genomes or transcriptomes of single cells in a massively parallel format.

[0045] In certain embodiments, nucleic acid sequence assay components that employ barcoding for labelling individual RNA (e.g., mRNA), cDNA, and DNA molecules, and / or for labeling for cell / well source (e.g., if wells pooled before sequencing analysis), and / or for labeling particular affixed entities (e.g., if droplet from two or more affixed entities are pooled prior to sequencing) are employed. Examples of such barcoding methodologies and reagents are found in Pat. Pub. US2007 / 0020640, Pat. Pub. 2012 / 0010091, U.S. Pat. 8,835,358, U.S. Pat. 8,481,292, Qiu et al. (Plant. Physiol., 133, 475-481, 2003), Parameswaran et al. (Nucleic Acids Res. 2007 Oct; 35(19): el30), Craig et al. reference (Nat. Methods, 2008, October, 5( 10): 887-893), Bontoux et al. (Lab Chip, 2008, 8:443-450), Esumi et al. (Neuro. Res., 2008, 60:439-451), Hug et al., J. Theor., Biol., 2003, 221:615-624), Sutcliffe et al. (PNAS, 97(5): 1976- 1981 ; 2000), Hollas and Schuler (Lecture Notes in Computer Science Volume 2812, 2003, pp 55-62), and W0201420127; all of which are herein incorporated by reference in their entireties, including for reaction conditions and reagents related to barcoding and sequencing of nucleic acids. In certain embodiments, the DropSeq method employing beads with primers attached to them arc employed to sequence nucleic acids from core- shell particles. An example of such a method is described in Macosko et al., Cell, 161 (5) : 1202- 1214 (see, e.g., Figure 1 therein), which is herein incorporated by reference in its entirety. In certain embodiments employing DropSeq, barcoded template switch oligos are bound to beads and oligo dT is supplied in solution along with RT PCR reagents. Reverse transcription (RT) can, for example, be performed as described in Kim et al., Anal Chem. 2018 Jan 16;90(2): 1273-1279, herein incorporated by reference. In other embodiments, barcoded oligo-dT beads are provided, the cells are lysed, mRNAs is captured on the beads, the emulsion is broken, and the drop is re-emulsified to capture mRNA beads with barcoded TSO beads where the TSO can be released by UV. Solution phase TSO can then be used for performing RT-PCR. Primers specific to the variable regions displayed on the surface of the SD cells can be employed to amplify such variable regions prior to sequencing.

[0046] In certain embodiments, unique oligo drops are provided to the fixed entities, and allow a link between imaging and genomics. For example, the unique oligos can contain two part 8-mer barcodes linked to polyA or TSO followed by 8-mer barcodes. In this regard, if one employs 96 barcoded oligos, selecting any three can generate 142,880 combinations. It is known what combination of three oligos are printed at each well position to identify that particular well. These oligos will also be sequenced and so when one sees a particular 3-oligo combination in the sequencing readouts, one knows the fixed entity and the image for that fixed entity.

[0047] In certain embodiments, the barcode tagging and sequencing methods of WO2014201273 (“SCRB-seq” method, herein incorporated by reference) are employed. The necessary reagents for the SCRB-seq method (e.g., modified as necessary for small volumes) are added to the aqueous droplets, each containing a lysed cell after the core-shell particles are formed. Briefly, the SCRB-seq method amplifies an initial mRNA sample from cells from a single fixed entity. Initial cDNA synthesis uses a first primer with: i) N6 for cell / well identification, ii) N10 for particular molecule identification, iii) a poly T stretch to bind mRNA, and iv) a region that creates a region where a second template- switching primer will hybridize. The second primer is a template switching primer with a poly G 3’ end, and 5’ end that has iso-bases. After cDNA amplification, the tagged cDNA single fixed entity samples are pooled. Then full-length cDNA synthesis occurs with two different primers, and full-length cDNA is purified. Next, a NEXTERA sequencing library is prepared using an i7 primer (adds one of 12 i7 tags to identify particular multi- well plates) and P5NEXTPT5 to add P5 tag for NEXTERA sequencing (P7 tag added to the other end for NEXTERA). The library is purified on a gel, and then NEXTERA sequencing occurs. As a non-liming example, with twelve i7 plate tags, and 384 cell / well- specific barcodes, this allows total of 4,608 single cell transciptomes to be done at once. This method allows for quantification of mRNA transcripts in single fixed entity.

[0048] In other embodiments, the barcode tagging and sequencing methods employ concepts from the Multi- seq method. For example, cells are incubated with anchor and co-anchor lipid modified oligonucleotides (LMO) and encapsulated in droplets. Individual barcodes in droplets and then formed core-shell particles can hybridize to exposed regions of the LMOs and these barcodes can be used instead of Drop-seq beads. Anchor-coanchor LMOs remain bound to individual cells at 4°C but can freely equilibrate between cells in a droplet at 37°C. Thus, a specific LMO-barcode combination can be used to link two cells in that droplet that can be tracked after emulsion breaking. In one example, a unique LMO-barcode combination can be randomly assembled in every microfluidic droplet. Barcodes may also be deterministically preprinted to a microwell array, and additionally provide linkage to imaging data recoded at specific micro well positions. In another embodiment, one cell in each combination may be LMO- barcoded before the combination in droplets. During incubation at 37°C, the LMO-barcodes will re-equilibrate to the initially non-barcoded cell and provide lasting information about coencapsulation. If a unbarcoded B-cell is combined with an LMO-barcoded antigen presenting cell (APC), this process will allow the type of APC to be read out by sequencing only the B-cell.

[0049] In practicing the methods of the present disclosure, one or more sorting steps may be employed. A sorting step sorts an aqueous droplet into one of two or more locations (e.g. into one of two or more fluid channels). In some cases, the sorting is into one of two fluid channels. Aqueous droplets are sorted based on one or more properties of the aqueous droplet or a component within the aqueous droplet. In addition, such sorting may either be passive sorting or active sorting. Active sorting includes the detection of one or more properties of an aqueous droplet, or a component within the aqueous droplet, and sorting based on the detected property. Passive sorting involves sorting an aqueous droplet without the active detection of a property. Sorting approaches of interest include, by are not necessarily limited to, approaches that involve the use of one or more sorting channels and one or more sorting elements. Sorting approaches which may be utilized in connection with the disclosed methods, systems and devices also include those described herein, and those described by Agrcsti, ct al., PNAS vol. 107, no 9, 4004-4009. For active sorting, the device includes one or more sorting elements and one or more detectors, wherein each detector is configured to detect one or more properties of an aqueous droplet, or a component within the aqueous droplet, and each sorting element is configured to sort the aqueous droplet into one of two or more locations based on the detecting by the detection element. In some cases, a sorting element is positioned in proximity to the sorting channel, such as an electrode in proximity to the sorting channel. In some cases, a sorting element is positioned within the sorting channel, such as a partial height flow divider in a sorting channel. In some cases, the device includes a sorting element positioned within the sorting channel and one or more sorting elements positioned in proximity to the sorting channel. Exemplary structures and methods for active sorting aqueous droplets are described in Cole et al., PNAS, 2017, 114, 33, 8728-8733; Clark et al., Lab Chip, 2018, 5, 18, 710-713; and Sciambi et al., Lab on a Chip, 2015, 15, 47-51, the disclosures of which are incorporated herein by reference for sorting elements.

[0050] A variety of different components can be included in the aqueous droplets to facilitate detection, including one or more fluorescent dyes (e.g., as part of oligonucleotide probe and / or to stain cell(s)). Such fluorescent dyes may be divided into families, such as fluorescein and its derivatives; rhodamine and its derivatives; cyanine and its derivatives; coumarin and its derivatives; Cascade Blue and its derivatives; Lucifer Yellow and its derivatives; BODIPY and its derivatives; and the like. Exemplary fluorophores include indocarbocyanine (C3), indodicarbocyanine (C5), Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Texas Red, Pacific Blue, Oregon Green 488, Alexa fluor-355, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor-555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, JOE, Lissamine, Rhodamine Green, BODIPY, fluorescein isothiocyanate (FITC), carboxy-fluorescein (FAM), phycoerythrin, rhodamine, dichlororhodamine (dRhodamine), carboxy tetramethylrhodamine (TAMRA), carboxy-X-rhodamine (ROX), LIZ, VIC, NED, PET, SYBR, PicoGreen, RiboGreen, and the like. Descriptions of fluorophores and their use, can be found in, among other places, R. Haugland, Handbook of Fluorescent Probes and Research Products, 9th ed. (2002), Molecular Probes, Eugene, Oreg.; M. Schena, Microarray Analysis (2003), John Wiley & Sons, Hoboken, N.J.; Synthetic Medicinal Chemistry 2003 / 2004 Catalog, Berry and Associates, Ann Arbor, Mich.; G. Hermanson, Bioconjugate Techniques, Academic Press (1996); and Glen Research 2002 Catalog, Sterling, VA. All publications and patents mentioned in the present application are herein incorporated by reference. Various modification and variation of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

CLAIMSWc claim:

1. A method comprising: a) generating an emulsion comprising a plurality of aqueous droplets in an oil phase such that at least some of said aqueous droplets are positive droplets that each comprise: i) an aqueous solution; ii) a gelling agent, iii) at least one cell, and iv) optionally a plurality of capture beads comprising: A) capture probes configured to hybridize to RNA from said cell, and optionally B) DNA capture sequences configured to hybridize to DNA from said cell; and optionally C) protein binding agents; b) treating said positive droplets in said emulsion such that said gelling agent gels in a plurality of said positive droplets thereby forming a plurality of core-shell gel particles that are present in said oil phase and that each comprise: i) an outer shell, and ii) an interior region, surrounded by said shell, that comprises: A) an interior solution that is at least partially aqueous, B) optionally an inner gel core; C) said at least one cell, and D) optionally at least some of said capture beads, wherein said treating optionally comprises cooling said positive droplets, and c) adding a lysing agent solution to said emulsion such that at least pail of said lysing solution agent migrates through said outer shell into said interior region and lyses said at least one cell releasing RNA and DNA molecules in each of said plurality of core-shell gel particles.

2. The method of claim 1, wherein said plurality of capture beads are present but without said DNA capture sequences, and the method further comprises: d) incubating said emulsion such that in each of said plurality of core-shell gel particles: A) at least some of said RNA molecules specifically hybridize to said capture probes thereby generating RNA-bound capture beads, and B) said DNA molecules are present in said interior solution and do not specifically hybridize to said capture probes, and do not migrate out of through said outer shell; andoptionally e) adding reverse-tran scription reagents to said emulsion to generate cDNA from said RNA bound to said RNA-bound capture beads.

3. The method of claim 2, further comprising: e) transferring at least some of said plurality of core-shell gel particles from said oil phase into an aqueous carrier, and f) treating said plurality of core-shell gel particles to generate a first sample and a second sample, wherein said first sample comprises said RNA-bound capture beads and is free or substantially free of said DNA molecules, and wherein said second sample comprises all or substantially all of said DNA molecules and is free or substantially free of said RNA molecules.

4. The method of claim 3, wherein said capture probes are attached to said capture beads via a reversible linker, and further comprising: g) treating said first sample such that said RNA molecules are reverse transcribed into cDNA either on said capture beads or after having been cleaved from said capture beads via said reversible linker.

5. The method of claim 4, further comprising: h) generating a sequencing library from said cDNA by attaching adapters to said cDNA or amplicons thereof and amplifying said cDNA and / or said amplicons using primers, wherein optionally said capture probes and / or said adapter comprise a barcode sequence, and i) sequencing said sequencing library to generate a plurality of RNA / cDNA sequencing reads.

6. The method of claim 3, further comprising: h) generating a sequencing library from said DNA molecules in said second sample, by attaching adapters to said DNA or amplicons thereof and amplifying said DNA molecules and / or said amplicons using primers, wherein optionally said adapters comprise a barcode sequence, and i) sequencing said sequencing library to generate a plurality of DNA sequencing reads.

7. The method of claim 1, wherein said plurality of capture beads are present with said DNA capture sequences, and the method further comprises: d) incubating said emulsion such that in each of said plurality of core-shell gel particles: A) at least some of said RNA molecules specifically hybridize to said capture probes, and B) at least some of said DNA moleculesspecifically or non-specifically hybridize to said DNA capture sequences; thereby generating RNA + DNA bound capture beads.

8. The method of claim 7, further comprise: e) transferring at least some of said plurality of core- shell gel particles from said oil phase into an aqueous carrier, and f) treating said plurality of core-shell gel particles to generate a first sample comprising purified or substantially purified RNA + DNA bound capture beads.

9. The method of claim 8, wherein said capture probes are attached to said capture beads via a reversible linker, and further comprising: g) at least one of the following:A) cleaving said RNA molecules from said RNA + DNA capture beads via said reversible linker to generate free RNA and DNA-capture beads, and separating said free RNA into a second sample and said DNA-capture beads into a third sample, and reverse transcribing said free RNA in said second sample into cDNA; orB) reverse transcribing said RNA on said RNA + DNA capture molecules into cDNA molecules, and cleaving said RNA and cDNA molecules from said RNA + DNA capture beads via said reversible linker to generate free RNA, free cDNA, and DNA-capture beads, and separating said free RNA and free cDNA into a second sample and said DNA-capture beads into a third sample.

10. The method of claim 9, further comprising: h) generating a first sequencing library from said cDNA from said second sample by attaching adapters to said cDNA or to amplicons thereof, and amplifying said cDNA and / or amplicons thereof using primers, wherein optionally said capture probes and / or said adapters each comprise a barcode sequence, and i) sequencing said first sequencing library to generate a plurality of RNA / cDNA sequencing reads.

11. The method of 9, further comprising: h) generating a second sequencing library from said DNA molecules in said third sample by attaching adapters to said DNA molecules or to amplicons thereof, and amplifying said DNA molecules and / or amplicons using primers, wherein optionally said capture sequences and / or said adapters each comprise a barcode sequence and i) sequencing said second sequencing library to generate a plurality of DNA sequencing reads.

12. The method of claim 1, wherein said plurality of capture beads arc not present, and the method further comprises: d) incubating said emulsion such that substantially all of said RNA molecules in each of said plurality of core-shell particles migrate out of said interior region through said outer shell and such that substantially all of said DNA molecules remain inside said outer shell in each of said core-shell particles.

13. The method of claim 12, further comprising: e) transferring at least some of said plurality of core-shell gel particles from said oil phase into an aqueous carrier, and f) treating said plurality of core-shell gel particles to generate a first sample comprising purified or substantially purified DNA molecules.

14. The method of claim 13, further comprising: f) generating a sequencing library from said DNA molecules in said first sample by attaching adapters to said DNA molecules or to amplicons thereof, and amplifying said DNA molecules and / or amplicons using primers, wherein optionally said adapters each comprise a barcode sequence and i) sequencing said sequencing library to generate a plurality of DNA sequencing reads.

15. The method of claim 1, wherein said at least one cell in each of said plurality of coreshell gel particles is one and only one cell.

16. The method of claim 1, wherein said generating said emulsion comprises either:1) combining an aqueous phase with said oil phase in bulk solution, wherein said aqueous phase comprises: i) an aqueous solution; ii) said gelling agent, iii) a plurality of cells, and iv) optionally a plurality of said capture beads, or2) the following steps: i) forming a first precursor emulsion comprising a plurality of first precursor aqueous droplets in an oil phase that each contain a precursor core-shell gel particle, ii) combining said plurality of first precursor aqueous droplets with a solution comprising cells to generate a second precursor emulsion comprising second precursoraqueous droplets in an oil phase that each contain at least one cell and a precursor coreshell gel particle, iii) heating said second precursor emulsion such that said precursor core-shell gel particle in each of said second precursor aqueous droplets melts forming liquid gel precursor, thereby forming said plurality of aqueous droplets recited in step a).

17. The method of claim 1, wherein said generating said emulsion comprises combining a first aqueous phase with said oil phase using a microfluidic droplet maker, wherein; I) said first aqueous phase comprises one or more of the following: i) an aqueous solution; ii) said gelling agent, iii) a plurality of cells, and iv) optionally a plurality of said capture beads, or II) wherein a second aqueous phase is combined with said first aqueous phase and said oil phase to generate said emulsion, and wherein said second aqueous phase comprises one or more of the following: i) an aqueous solution; ii) said gelling agent, iii) a plurality of cells, and iv) optionally a plurality of said capture beads.

18. The method of claim 17, wherein said microfluidic droplet maker comprises a co-flow droplet maker, and optionally comprises: a How focusing component, a T-junction, a step emulsification component, and / or a jet triggered droplet generator.

19. The method of claim 1, wherein said gelling agent comprises chitosan.

20. The method of claim 17, wherein said microfluidic droplet maker employs particle templated emulsification or geometrically mediated breakup in a hierarchical array and optionally without requiring the substantial control of flow rate.

21. The method of claim 1, wherein said positive droplets further comprise cell media.

22. The method of claim 1, wherein said positive droplets comprise at least to cells, or only two cells.

23. The method of claim 1 , wherein said positive droplets have a diameter of from about 1 pm to 1000 pm.

24. The method of claim 1, wherein said lysing agent solution comprises a lysing agent, wherein said lysing agent is optionally a protease.

25. The method of claim 1, wherein said lysing agent solution comprises a detergent or other chemical that overcomes inhibition and optionally facilitate barcoding of nucleic acids and / or micelle transport.

26. The method of any of claims 5, 6, 10, 11, and 14 wherein said amplifying is performed by PCR or multiple displacement amplification (MDA).

27. The method of any of claims 5, 6, 10, 11, and 14, wherein said sequencing is performed by a method selected from: Ab-seq, CITE-seq, sequencing by synthesis, and next generation sequencing.

28. The method of any of claims 1-27, wherein said plurality of capture beads are present and comprise said protein binding agents.

29. The method of claim 28, wherein said protein binding agents bind target proteins from said at least one cell, and optionally wherein said target proteins are purified into a purified protein sample.

30. The method of claim 1, wherein said emulsion is a single emulsion or double emulsion.

31. The method of claim 30, further comprising: sorting said plurality of core-shell gel particles based on the presence or absence of a signal or detectable molecule from each of said plurality of core-shell gel particles.

32. The method of claim 31 , wherein said sorting is based on the presence or absence of said detectable molecule arc particular barcoded or unbarcodcd DNA or mRNA molecules.

33. A composition, system, or kit comprising: a) a plurality of core-shell gel particles that each comprise: i) an outer gel shell, and ii) an interior region, surrounded by said shell, that comprises: A) an interior solution that is at least partially aqueous, B) optionally an inner gel core; C) said at least one cell, and D) a plurality of capture beads comprising: I) capture probes configured to hybridize to RNA from said cell, and optionally II) DNA capture sequences configured to hybridize to DNA from said cell; and optionally III) protein binding agents; and b) optionally a lysing agent solution.

34. The composition, system, or kit of claim 33, further comprising an aqueous solution or an oil carrier phase, and wherein said plurality of core-shell gel particles are present in said aqueous solution or said oil carrier phase.

35. The method, composition, system, or kit of any of the above claims, wherein the RNA comprising mRNA.

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