Compositions, methods and systems for forming beads using improved polymers

Abstract

The present disclosure provides systems and methods for preparing hydrogels, the hydrogels comprising cells, cell nuclei, or one or more components derived from cells or cell nuclei. A method for preparing a hydrogel may include: providing cells or cell nuclei, a first polymer, wherein the first polymer comprises a plurality of first cross-linking precursors, each of the plurality of first cross-linking precursors comprising an azide group; providing a second polymer, wherein the second polymer comprises a plurality of second cross-linking precursors, each of the plurality of second cross-linking precursors comprising an alkyne group; and cross-linking the first polymer and the second polymer by a reaction between a first portion of the first cross-linking precursor and a second portion of the second cross-linking precursor, thereby providing the hydrogel comprising the cells or cell nuclei.

Description

Technical Field

[0001] The present disclosure relates to methods, compositions and systems for preparing hydrogels comprising cells, cell nuclei or one or more components derived from cells or cell nuclei. Background Art

[0002] Samples can be processed for various purposes such as identifying the type of a portion of the sample within the sample. The sample can be a biological sample. Biological samples can be processed for various purposes such as detecting a disease (e.g., cancer) or identifying a particular species. There are various methods for processing samples such as polymerase chain reaction (PCR) and sequencing.

[0003] Biological samples can be processed in various reaction environments, such as partitions. The partitions can be wells or droplets. The biological samples can be processed using droplets or wells in a manner that allows the biological samples to be separated and processed individually. For example, such droplets can be isolated from other droplet fluids, so that the corresponding environment in the droplet can be accurately controlled.

[0004] The biological samples in the partitions can be subjected to various processes such as chemical processes or physical processes. The samples in the partitions can be subjected to heating or cooling or chemical reactions, such as producing species that can be qualitatively or quantitatively processed. Summary of the invention

[0005] Hydrogel matrix (comprising beads) can create a semi-open system, and the semi-open system can encapsulate macromolecules in the matrix boundary while allowing small molecules to penetrate the matrix. Macromolecules can be biological samples, including cells, large proteins or long nucleic acids. Small molecules can be reagents, smaller proteins or shorter nucleic acids, for example. For example, enzymes can be small enough to penetrate the matrix. The hydrogel matrix can also include unstable bonds so that after the hydrogel matrix is ​​degraded, the encapsulated macromolecules can be released into the surrounding environment from the scope of the matrix. Provided herein are methods, systems and compositions for producing hydrogel matrix, which can encapsulate macromolecules and allow small molecules to penetrate the matrix.

[0006] In some aspects, the present disclosure provides a gel comprising: (a) cells, cell nuclei or one or more components derived from cells; (b) two or more polymers; and (c) a plurality of joints, each of the plurality of joints comprising a 1,2,3-triazole moiety, wherein the joint cross-links the two or more polymers. In some embodiments, each of the two or more polymers independently comprises at least one selected from the group consisting of: polyolefins, olefin copolymers, acrylic acid, vinyl polymers, polyesters, polycarbonates, polyamides, polyimides, formaldehyde resins, polyurethanes, ether polymers, cellulose, thermoplastic elastomers, and thermoplastic polyurethanes. In some embodiments, each of the two or more polymers is independently polyacrylamide. In some embodiments, each of the plurality of joints is independently connected to the amide of the two or more polymers. In some embodiments, each of the plurality of joints comprises an unstable bond. In some embodiments, the unstable bond is a chemically unstable bond, a thermally unstable bond, or a photolabile bond. In some embodiments, the unstable bond comprises a disulfide bond. In some embodiments, the 1,2,3-triazole moiety is formed by treating an azide group with an alkyne group under conditions sufficient to form the 1,2,3-triazole moiety. In some embodiments, the gel further comprises at least one reagent encapsulated within the gel.

[0007] In some embodiments, the gel is a hydrogel. In some embodiments, the gel further comprises a charged species. In some embodiments, the charged species is positively charged. In some embodiments, the charged species comprises trimethylammonium. In some embodiments, the charged species is negatively charged. In some embodiments, the charged species comprises phosphate. In some embodiments, the charged species is connected to the polymer or gel network. In some embodiments, at least one of the two or more polymers is a charged polymer. In some embodiments, the charged polymer is a positively charged polymer. In some embodiments, the positively charged polymer comprises chitosan or polyethyleneimine. In some embodiments, the charged polymer is a negatively charged polymer. In some embodiments, the negatively charged polymer comprises alginate. In some embodiments, at least one of the two or more polymers comprises a charged moiety, and wherein the charged moiety is connected to the at least one of the two or more polymers by a joint. In some embodiments, the joint comprises an unstable bond capable of cutting the charged moiety from the at least one of the two or more polymers. In some embodiments, the unstable bond is a chemically unstable bond, a thermally unstable bond, or a photolabile bond.

[0008] In some aspects, the present disclosure provides a method for forming a gel including a cell or a cell nucleus, the method comprising: (a) providing (i) a first polymer, wherein the first polymer includes a plurality of first cross-linking precursors, each of the plurality of first cross-linking precursors including an azide group; (ii) a second polymer, wherein the second polymer includes a plurality of second cross-linking precursors, each of the plurality of second cross-linking precursors including an alkyne group; and (iii) the cell or the cell nucleus; (b) cross-linking the first polymer and the second polymer by a reaction between a first portion of the first cross-linking precursor and a second portion of the second cross-linking precursor, thereby forming the gel including the cell or the cell nucleus. In some embodiments, the first polymer and the second polymer independently include at least one selected from the group consisting of: polyolefins, olefin copolymers, acrylic acid, vinyl polymers, polyesters, polycarbonates, polyamides, polyimides, formaldehyde resins, polyurethanes, ether polymers, cellulose, thermoplastic elastomers, and thermoplastic polyurethanes. In some embodiments, the first polymer or the second polymer further includes an unstable bond. In some embodiments, the first polymer and the second polymer further include an unstable bond. In some embodiments, the unstable bond is a disulfide bond. In some embodiments, at least about 80% of the labile bonds remain intact during the reaction in (b). In some embodiments, the reaction forms a 1,2,3-triazole between the azide and the alkyne. In some embodiments, the method further comprises, prior to (b), providing a catalyst configured to catalyze the reaction in (b). In some embodiments, the method further comprises, after (b), removing the catalyst and / or its derivative from the gel. In some embodiments, the gel is formed from a plurality of the first polymers and a plurality of the second polymers. In some embodiments, the gel is a hydrogel.

[0009] In some embodiments, the method further comprises, after (b), lysing the cell or the cell nucleus to release one or more cell or cell nucleus components into the gel. In some embodiments, the one or more cell or cell nucleus components include nucleic acids. In some embodiments, the nucleic acids include ribonucleic acids. In some embodiments, the ribonucleic acids are messenger ribonucleic acids (mRNA). In some embodiments, the ribonucleic acids are microribonucleic acids (miRNA). In some embodiments, the nucleic acids include deoxyribonucleic acids (DNA). In some embodiments, the DNA is genomic DNA. In some embodiments, the one or more cell or cell nucleus components include chromatin. In some embodiments, the one or more cell or cell nucleus components include proteins. In some embodiments, the one or more cell or cell nucleus components can be retained in the gel. In some embodiments, the one or more cell or cell nucleus components can be retained in the gel for at least 1, at least 2, at least 3, at least 4, at least 5, at least 12 or at least 24 hours. In some embodiments, the method further comprises denaturing the DNA. In some embodiments, the denaturation comprises contacting the gel with a chemical reagent. In some embodiments, the chemical reagent is an alkaline reagent. In some embodiments, the method further comprises permeabilizing the cell or the cell nucleus after (b). In some embodiments, the method further comprises, prior to (b), separating the first polymer, the second polymer, and the cell or cell nucleus together into partitions. In some embodiments, the partitions are pores. In some embodiments, the partitions are aqueous droplets in an emulsion. In some embodiments, the partitions comprise a reagent configured to catalyze the reaction in (b). In some embodiments, the emulsion comprises an oil phase, the oil phase comprises a reagent, the reagent comprises a copper (II) portion, and wherein the aqueous droplets comprise a reducing agent capable of reducing the copper (II) portion to a copper (I) portion, wherein the copper (I) portion catalyzes the reaction in (b). In some embodiments, the emulsion comprises a reagent that facilitates the transport of the copper (II) portion from the oil phase to the aqueous droplets. In some embodiments, the method further comprises, prior to (b), lysing or permeabilizing the cells or cell nuclei in the partitions.

[0010] In some aspects, the present disclosure provides a method for generating cell beads, the method comprising: (a) generating a partition including cells from a plurality of cells or cell nuclei from a plurality of cell nuclei, a polymer or gel precursor, and a charged species; and (b) subjecting the partition to conditions sufficient to react the polymer or gel precursor to generate a polymer or gel network, the polymer or gel network including (i) the cell or its derivative, and (ii) the charged species, thereby providing the cell beads. In some embodiments, the partition is among a plurality of partitions. In some embodiments, the method further comprises generating a plurality of cell beads from the plurality of partitions. In some embodiments, the charged species is positively charged. In some embodiments, the charged species comprises trimethylammonium. In some embodiments, the charged species is (3-acrylamidopropyl) trimethylammonium. In some embodiments, the charged species is negatively charged. In some embodiments, the charged species comprises phosphate. In some embodiments, the charged species is connected to the polymer or gel network. In some embodiments, the cell beads include a plurality of chemical crosslinkers. In some embodiments, the charged species is connected to a chemical crosslinker of the chemical crosslinker. In some embodiments, the method further comprises, prior to (b), subjecting the cell bead to conditions sufficient to lyse the cell or cell nucleus to release one or more cellular or cell nuclear components into the cell bead.

[0011] In some embodiments, the method further includes, after (b), subjecting the cell beads to conditions sufficient to lyse the cells or nuclei to release one or more cell or nucleus components into the cell beads. In some embodiments, the one or more cell or nucleus components include nucleic acids. In some embodiments, the nucleic acids include ribonucleic acids. In some embodiments, the ribonucleic acids are messenger RNAs. In some embodiments, the nucleic acids include deoxyribonucleic acids. In some embodiments, the one or more cell or nucleus components include proteins. In some embodiments, the one or more cell or nucleus components can be retained in the cell beads. In some embodiments, the one or more cell or nucleus components can be retained in the cell beads for at least 1, at least 2, at least 3, at least 4, at least 5, at least 12, or at least 24 hours.

[0012] In some aspects, the present disclosure provides a method for generating cell beads, the method comprising: (a) generating a partition including (i) cells from multiple cells or cell nuclei from multiple cell nuclei and (ii) a charged polymer or gel precursor; and (b) subjecting the partition to conditions sufficient to react the polymer or gel precursor to generate a charged polymer or gel network, the charged polymer or gel network including the cell or the cell nucleus or a derivative thereof, thereby providing the cell beads. In some embodiments, the charged polymer or gel precursor includes a positive charge. In some embodiments, the charged polymer or gel precursor includes chitosan. In some embodiments, the charged polymer or gel precursor includes polyethyleneimine. In some embodiments, the charged polymer or gel precursor includes a negative charge. In some embodiments, the charged polymer or gel precursor includes alginate. In some embodiments, the method further includes, before (b), subjecting the cell beads to conditions sufficient to lyse the cells or cell nuclei to release one or more cell or cell nuclear components into the cell beads. In some embodiments, the method further includes, after (b), subjecting the cell bead to conditions sufficient to lyse the or nucleus to release one or more cell or nucleus components into the cell bead. In some embodiments, the one or more cell or nucleus components nucleic acid. In some embodiments, the nucleic acid comprises ribonucleic acid. In some embodiments, the ribonucleic acid is messenger ribonucleic acid. In some embodiments, the nucleic acid comprises deoxyribonucleic acid. In some embodiments, the one or more cell or nucleus components comprise protein. In some embodiments, the one or more cell or nucleus components can be retained in the cell bead. In some embodiments, the one or more cell or nucleus components can be retained in the cell bead for at least 1, at least 2, at least 3, at least 4, at least 5, at least 12 or at least 24 hours.

[0013] In some aspects, the present disclosure provides a composition for analyzing one or more components from a cell, the composition comprising a cell bead, the cell bead comprising a polymerized or cross-linked polymer network, which comprises a cell, a nucleus, or one or more components derived from a cell or a nucleus, wherein the polymerized or cross-linked polymer network is charged. In some embodiments, the polymer network is positively charged. In some embodiments, the polymer network comprises polyethyleneimine. In some embodiments, the polymer network comprises chitosan. In some embodiments, the polymer network is negatively charged. In some embodiments, the polymer network comprises alginate.

[0014] In some aspects, the present disclosure provides a composition for analyzing one or more components from a cell, the composition comprising a cell bead, the cell bead comprising a polymerized or cross-linked polymer network, which includes (i) a cell, a cell nucleus, or one or more components derived from a cell or a cell nucleus; and (ii) a charged species. In some embodiments, the polymer network includes a chemical crosslinker. In some embodiments, the cell bead includes a component from the cell connected to the chemical crosslinker. In some embodiments, the charged species is connected to the polymer network. In some embodiments, the charged species is covalently connected to the polymer network. In some embodiments, the charged species is connected to a component from the cell. In some embodiments, the charged species is non-covalently connected to the component from the cell. In some embodiments, the charged species is positively charged. In some embodiments, the charged species includes trimethylammonium. In some embodiments, the charged species is (2-aminoethyl) trimethylammonium. In some embodiments, the charged species is (3-acrylamidopropyl) trimethylammonium. In some embodiments, the charged species is negatively charged. In some embodiments, the charged species includes phosphate.

[0015] For those skilled in the art, additional aspects and advantages of the present disclosure will become apparent through the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other different embodiments, and its several details can be modified in various obvious aspects, all without departing from the present disclosure. Therefore, the drawings and description will be regarded as illustrative rather than restrictive in nature.

[0016] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description setting forth illustrative embodiments, in which the principles of the present invention are utilized, and in the accompanying drawings (also referred to herein as "Figures / FIG."):

[0018] Figure 1 An example of a microfluidic channel structure for separation of single bioparticles is shown.

[0019] Figure 2 An example of a microfluidic channel structure for delivering barcode-carrying beads to droplets is shown.

[0020] Figure 3 An example of a microfluidic channel structure for co-separation of biological particles and reagents is shown.

[0021] Figure 4 An example of a microfluidic channel structure for controlled separation of beads into discrete droplets is shown.

[0022] Figure 5 Examples of microfluidic channel structures for increasing droplet generation throughput are shown.

[0023] Figure 6 Another example of a microfluidic channel structure for increasing droplet generation throughput is shown.

[0024] Figure 7 An example of a microfluidic channel structure for co-separating biological particles and reagents to form droplets configured to form a hydrogel is shown.

[0025] Figure 8 Example hydrogel compositions and steps for forming hydrogels in droplets are shown.

[0026] Fig. 9 An example method for generating click chemistry polymer networks is shown.

[0027] Fig.10 An exemplary method for producing the cell beads of the present disclosure is presented.

[0028] Fig.11A -B shows an example charged hydrogel polymer network. Fig.11A A schematic diagram of a cell bead comprising a positively charged species attached to a polymer network is shown. Fig. 11B A schematic diagram of a cell bead comprising a negatively charged species attached to a polymer network is shown.

[0029] Fig. 12A -B shows another example charged hydrogel polymer network. Fig. 12A A schematic diagram of a cell bead comprising a positively charged species linked to a disulfide-containing chemical cross-linker is shown. Fig. 12B A schematic diagram of a cell bead comprising a negatively charged species linked to a disulfide-containing chemical cross-linker is shown.

[0030] Fig.13 An example process for generating droplets including components from cells is shown.

[0031] Fig.14Another example process for generating droplets including components from cells is shown.

[0032] Fig.15 An example process for generating cell beads comprising complementary deoxyribonucleic acid is shown.

[0033] Fig.16A An example method for generating droplets including barcoded beads and cell beads is schematically illustrated. Fig. 16B An example microfluidic architecture for generating cell beads is shown. Fig. 16C An example microfluidic architecture for generating droplets including barcoded beads and cell beads is shown. Fig.16D Demonstrated the use of Fig. 16C The architecture shown in FIG. 5 is an example droplet generation process for generating droplets including barcoded beads and cell beads.

[0034] Fig.17 An example process for generating cell beads and partitions including cell beads and gel beads is shown.

[0035] Fig.18 An example computer system is shown that is programmed or otherwise configured to implement the methods and systems provided herein.

[0036] Fig.19 Representative microscopy images of cell beads encapsulating cells are shown.

[0037] Fig. 20 Shown are the results of an experiment sequencing DNA obtained from cells included in cell beads.

[0038] Fig.21A An example workflow for generating cell beads is illustrated. Fig. 21B Shown are imaging results of cell beads generated without cell centering. Fig. 21C Shown are the imaging results of cell beads generated with cell centering.

[0039] Fig. 22 A graph showing the results of a cell centering experiment from Example 4 is shown.

[0040] Fig.23 A graph showing the results of a cell bead generation experiment from Example 5 is shown.

[0041] Fig.24 A graph showing the results of a cell bead generation experiment from Example 6 is shown.

[0042] Fig.25A Shown are results from the cell bead generation experiments described in Example 7 involving the use of different sodium ascorbate concentrations. Fig.25BResults from the cell bead generation experiments described in Example 7 involving the use of different gelation times are shown. Fig.25C Shown are results from the cell bead generation experiments described in Example 7 involving the use of different THPTA concentrations. Fig.25D Shown are results from the cell bead generation experiments described in Example 7 involving the use of different sodium ascorbate concentrations.

[0043] Fig.26A Results from the cell bead generation experiments described in Example 7 involving the use of different CuAcAc concentrations are shown. Fig.26B Results from the cell bead generation experiments described in Example 7 involving the use of different gelation times are shown.

[0044] Fig.27A Shown are results from the cell bead generation experiments described in Example 8 involving the use of different THPTA concentrations. Fig.27B Shown are results from the cell bead generation experiments described in Example 8 involving the use of different sodium ascorbate concentrations. Fig.27C Shown are results from the cell bead generation experiments described in Example 8 involving the use of different sodium ascorbate concentrations. Fig.27D Shown are results from the cell bead generation experiments described in Example 8 involving the use of different sodium ascorbate concentrations.

[0045] Fig.28A Shown are results from the cell bead generation experiments described in Example 8 involving the use of different THPTA concentrations. Fig.28B Shown are results from the cell bead generation experiments described in Example 8 involving the use of different gelation times.

[0046] Fig.29 An exemplary low copper concentration click chemistry cross-linking reaction using an azide-picolyl modified linker is shown.

[0047] Fig.30 Exemplary copper-free click chemistry cross-linking reactions using azide-modified and DBCO-modified linkers are shown.

[0048] Fig.31A Shown are the results of low copper concentration click chemistry cross-linking using an azide-picolyl modified linker as described in Example 11.

[0049] 31B shows the results of low copper concentration click chemistry cross-linking using an azide-picolyl modified linker as described in Example 11.

[0050] Fig.32An exemplary click chemistry cross-linking reaction is shown to form cross-links with labile carbamate bonds, and subsequent cleavage of the carbamate with DETA and heat.

[0051] Fig.33 Results for crosslinking with DETA and thermally degradable urethane as described in Example 12 are shown.

[0052] Fig.34A An exemplary propargyl-polypeptide linker that can form a protease-cleavable cross-link via click chemistry is shown.

[0053] Fig.34B The formation of exemplary protease-cleavable polypeptide cross-links by click chemistry and subsequent selective protease-catalyzed degradation of the cross-links are shown.

[0054] Fig.35 An exemplary reaction to form cross-linked polymers by click chemistry is shown, wherein one of the polymers is modified with a poly-T oligonucleotide capture agent attached by click chemistry. DETAILED DESCRIPTION

[0055] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided as examples only. Various changes, modifications and substitutions may be envisioned by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0056] Where values ​​are described as ranges, it should be understood that such disclosure includes disclosure of all possible sub-ranges within such ranges, as well as specific values ​​falling within such ranges, whether or not a specific value or specific sub-range is expressly stated.

[0057] The term "barcode" as used herein generally refers to a label or identifier that conveys or is capable of conveying information about an analyte. A barcode can be part of an analyte. A barcode can be independent of the analyte. In addition to the endogenous characteristics of the analyte (e.g., the size of the analyte or terminal sequence), a barcode can be a label or combination of labels that is connected to an analyte (e.g., a nucleic acid molecule). A barcode can be unique. A barcode can have a variety of different formats. For example, a barcode can include: a polynucleotide barcode; a random nucleic acid and / or amino acid sequence; and a synthetic nucleic acid and / or amino acid sequence. A barcode can be connected to an analyte in a reversible or irreversible manner. A barcode can be added to a fragment of, for example, a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before, during, and / or after sample sequencing. A barcode can identify and / or quantify a single sequencing read in real time.

[0058] As used herein, the term "real time" may refer to a response time of less than about 1 second, one tenth of a second, one hundredth of a second, milliseconds, or less. The response time may be greater than 1 second. In some cases, real time may refer to simultaneous or substantially simultaneous processing, detection, or identification.

[0059] As used herein, the term "subject" generally refers to an animal, such as a mammal (e.g., a human) or avian (e.g., a bird) or other organisms such as a plant. The subject can be a vertebrate, a mammal, a rodent (e.g., a mouse), a primate, a monkey, or a human. Animals can include, but are not limited to, farm animals, competitive animals, and pets. The subject can be a healthy or asymptomatic individual, an individual suffering from or suspected of having a disease (e.g., cancer) or a disease tendency, and / or an individual in need of therapy or suspected of needing therapy. The subject can be a patient.

[0060] As used herein, the term "genome" generally refers to genomic information from a subject, which can be, for example, at least a portion or all of a subject's genetic information. The genome can be encoded with DNA or RNA. The genome can include coding regions (for example, encoding proteins) and non-coding regions. The genome can include the sequences of all chromosomes in an organism. For example, the human genome generally has 46 chromosomes. All of these sequences can constitute the human genome.

[0061] The terms "adapter", "adapter" and "tag" can be used synonymously. An adaptor or tag can be coupled to the polynucleotide sequence to be "labeled" by any method, including ligation, hybridization or other methods.

[0062] As used herein, the term "sequencing" generally refers to methods and techniques for determining the sequence of nucleotide bases in one or more polynucleotides. A polynucleotide can be, for example, a nucleic acid molecule such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single-stranded DNA). Sequencing can be performed by a variety of currently available systems, such as, but not limited to, Pacific Biosciences OXFORD or Life Technologies (ION ) sequencing system. Alternatively or in addition, nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR or real-time PCR) or isothermal amplification can be used for sequencing. Such systems can provide a plurality of original genetic data corresponding to the genetic information of a subject (e.g., a person), as generated by the system from a sample provided by the subject. In some instances, such systems provide sequencing reads (also referred to herein as "reads"). The reads can include a string of nucleic acid bases corresponding to a sequence of nucleic acid molecules that have been sequenced. In some cases, the systems and methods provided herein can be used together with proteomics information.

[0063] As used herein, the term "bead" generally refers to a particle. A bead may be a solid or semisolid particle. A bead may be a gel bead. A gel bead may contain a polymer matrix (e.g., a matrix formed by polymerization or cross-linking). A bead may be formed of a polymeric material. A bead may be magnetic or non-magnetic. A bead may be rigid. A bead may be flexible and / or compressible. A bead may be destructible or soluble.

[0064] As used herein, the term "sample" generally refers to a biological sample of a subject. A biological sample can include any number of macromolecules, such as cellular macromolecules. A biological sample can be a nucleic acid sample or a protein sample. A biological sample can also be a carbohydrate sample or a lipid sample. A biological sample can be derived from another sample. A sample can be a tissue sample such as a biopsy, a core biopsy, a needle aspirate, or a fine needle aspirate. A sample can be a fluid sample such as a blood sample, a urine sample, or a saliva sample. A sample can be a skin sample. A sample can be a cheek swab. A sample can be a plasma or serum sample. A sample can be a cell-free or cell-free sample. A cell-free sample can contain extracellular polynucleotides. Extracellular polynucleotides can be separated from a body sample, and the body sample can be selected from the group consisting of: blood, plasma, serum, urine, saliva, mucosal excretions, sputum, feces, and tears.

[0065] As used herein, the term "biological particle" generally refers to a discrete biological system derived from a biological sample. A biological particle can be a virus. A biological particle can be a cell or a derivative of a cell. A biological particle can be an organelle. A biological particle can be a rare cell from a cell population. A biological particle can be any type of cell, including but not limited to prokaryotic cells, eukaryotic cells, bacteria, fungi, plants, mammalian or other animal cell types, mycoplasmas, normal tissue cells, tumor cells, or any other cell type, whether derived from a unicellular or multicellular organism. A biological particle can be or can include a matrix (e.g., a gel or polymer matrix) comprising a cell or one or more components from a cell (e.g., a cell bead), such as DNA, RNA, organelles, proteins, or any combination thereof from a cell. A biological particle can be obtained from a subject's tissue. A biological particle can be a hardened cell. Such hardened cells may or may not include a cell wall or a cell membrane. A biological particle can include one or more components of a cell, but may not include other components of a cell. Examples of such components are a nucleus or an organelle. A cell can be a living cell. Living cells may be capable of being cultured, for example, when enclosed in or when comprising a gel or polymer matrix.

[0066] As used herein, the term "macromolecular component" generally refers to a macromolecule contained within a bioparticle. A macromolecular component may include a nucleic acid. A macromolecular component may include a DNA. A macromolecular component may include an RNA. The RNA may be a messenger RNA (mRNA), a ribosomal RNA (rRNA), or a transfer RNA (tRNA). The RNA may be a transcript. A macromolecular component may include a protein. A macromolecular component may include a peptide. A macromolecular component may include a polypeptide.

[0067] As used herein, the term "molecular tag" generally refers to a molecule that can bind to a macromolecular component. A molecular tag can bind to a macromolecular component with high affinity. A molecular tag can bind to a macromolecular component with high specificity. A molecular tag can include a nucleotide sequence. A molecular tag can include a nucleic acid sequence. A nucleic acid sequence can be at least a portion or all of a molecular tag. A molecular tag can be a nucleic acid molecule or can be a portion of a nucleic acid molecule. A molecular tag can be an oligonucleotide or a polypeptide. A molecular tag can include a DNA aptamer. A molecular tag can be or include a primer. A molecular tag can be or include a protein. A molecular tag can include a polypeptide. A molecular tag can be a barcode.

[0068] Provided herein are compositions and methods for forming hydrogel matrices (including beads) by emulsion gelation. In addition, the hydrogel matrix can encapsulate macromolecules such as biological samples. The biological sample can be a cell, a large protein, or a long nucleic acid. The hydrogel matrix can include cells or one or more components from cells (e.g., cell beads). The hydrogel matrix can allow smaller molecules to penetrate the matrix. Smaller molecules can be reagents, smaller proteins, or shorter nucleic acids. Smaller proteins can be enzymes. The hydrogel can be degradable.

[0069] In one aspect, the present disclosure provides a composition of a degradable hydrogel, the composition comprising two or more polymers and a plurality of linkers configured to form crosslinks. Each of the plurality of linkers may comprise an unstable bond and a 1,2,3-triazole moiety. The two or more polymers may be crosslinked via such linkers.

[0070] In one aspect, the present disclosure provides a method for forming a hydrogel. The method may include (a) providing a first polymer, wherein the first polymer includes a plurality of first cross-linking precursors; (b) providing a second polymer, wherein the second polymer includes a plurality of second cross-linking precursors; and (c) cross-linking the first polymer and the second polymer by a reaction between a first portion of the first cross-linking precursor and a second portion of the second cross-linking precursor, thereby forming the hydrogel.

[0071] In one aspect, the systems and methods described herein provide for compartmentalization, deposition or separation of the macromolecular constituent contents of a single bioparticle into discrete compartments or partitions (interchangeably referred to herein as partitions), wherein each partition maintains its own contents separate from the contents of other partitions. A partition may be a droplet in an emulsion. A partition may include one or more other partitions.

[0072] Partitions of the present disclosure may include bioparticles and / or their macromolecular components. Partitions may include one or more gel beads. Partitions may include one or more cell beads. Partitions may include single gel beads, single cell beads, or both single cell beads and single gel beads. Cell beads may be bioparticles and / or their one or more macromolecular components, which are wrapped inside a gel or polymer matrix, such as by polymerization of droplets containing bioparticles and precursors capable of polymerization or gelation. Unique identifiers such as bar codes may be injected into droplets before, after, or simultaneously with droplet generation, such as by microcapsules (e.g., beads), as further described below. Microfluidic channel networks (e.g., on a chip) may be used to generate partitions as described herein. Alternative mechanisms may also be employed in the separation of single bioparticles, including porous membranes, through which an aqueous mixture of cells is extruded into a non-aqueous fluid.

[0073] Partitions can flow within a fluid stream. Partitions can include, for example, microvesicles with an external barrier around an internal fluid center or core. In some cases, partitions can include a porous matrix capable of entraining and / or retaining materials within its matrix. Partitions can include aqueous fluid droplets within a non-aqueous continuous phase (e.g., an oil phase). Partitions can include droplets of a first phase within a second phase, wherein the first phase and the second phase are immiscible. For example, a variety of different containers are described in U.S. Patent Application Publication No. 2014 / 0155295, which is incorporated herein by reference in its entirety for all purposes. For example, an emulsion system for producing stable droplets in a non-aqueous or oil continuous phase is described in U.S. Patent Application Publication No. 2010 / 0105112, which is incorporated herein by reference in its entirety for all purposes.

[0074] In the case of droplets in emulsions, in a non-limiting example, the distribution of a single biological particle to a discrete partition can be achieved by introducing the flow of the biological particles in the aqueous fluid into the flow of the non-aqueous fluid, so that droplets are produced at the junction of the two streams. By providing water flow with a certain concentration and / or biological particle flow rate, the occupancy rate of the resulting partition (for example, the number of biological particles in each partition) can be controlled. In the case of using a single biological particle partition, the relative flow rate of the immiscible fluid can be selected so that the partition can contain less than one biological particle on average per partition to ensure that those occupied partitions are mainly single occupied. In some cases, the partition between multiple partitions can contain at most one biological particle (for example, beads, cells or cell materials). In certain embodiments, the relative flow rate of the fluid can be selected so that most of the partitions are occupied, for example, only a small percentage of unoccupied partitions are allowed. Streams and channel architectures can be controlled to ensure a given number of single occupied partitions, unoccupied partitions less than a certain level, and / or multiple occupied partitions less than a certain level.

[0075] Figure 1An example of a microfluidic channel structure 100 for separating single biological particles is shown. The channel structure 100 can include channel segments 102, 104, 106, and 108 connected at a channel junction 110. In operation, a first aqueous fluid 112 containing suspended biological particles (or cells) 114 can be transported along the channel segment 102 to the junction 110, and a second fluid 116 that is immiscible with the aqueous fluid 112 is delivered from each of the channel segments 104 and 106 to the junction 110 to produce discrete droplets 118, 120 of the first aqueous fluid 112 that flow into the channel segment 108 and flow out of the junction 110. The channel segment 108 can be fluidically coupled to an outlet reservoir that can store and / or harvest the discrete droplets. The generated discrete droplets can contain a single biological particle 114 (such as droplet 118). The generated discrete droplets can contain more than one single biological particle 114 (such as droplet 118). Figure 1 14). The discrete droplets may not contain biological particles 114 (such as droplet 120). Each discrete partition may keep its own contents (e.g., a single biological particle 114) separate from the contents of other partitions.

[0076] The second fluid 116 may include an oil such as a fluorinated oil that includes a fluorosurfactant for stabilizing the resulting droplets, e.g., inhibiting subsequent coalescence of the resulting droplets 118, 120. Examples of particularly useful separation fluids and fluorosurfactants are described, for example, in U.S. Patent Application Publication No. 2010 / 0105112, which is incorporated herein by reference in its entirety for all purposes.

[0077] It should be understood that the passage section described herein can be coupled to any one of a variety of different fluid sources or receiving components, including fluid components of reservoirs, pipes, manifolds or other systems.It should be understood that microfluidic channel structure 100 can have other geometric shapes.For example, the microfluidic channel structure can have more than one channel junction.For example, the microfluidic channel structure can have 2, 3, 4 or 5 passage sections, and each passage section carries biological particles, cell beads and / or gel beads that meet at the channel junction.Fluid can flow along one or more channels or reservoirs via one or more fluid flow units.Fluid flow units can include compressors (for example, positive pressure is provided), pumps (for example, negative pressure is provided), actuators, etc. to control the flow of fluid.Fluid can also or otherwise be controlled by the pressure difference, centrifugal force, electric pumps, vacuum, capillaries or gravity flow, etc. applied.

[0078] The generated droplets may include two subsets of droplets: (1) occupied droplets 118, which contain one or more biological particles 114 and (2) unoccupied droplets 120, which do not contain any biological particles 114. The occupied droplets 118 may include a single occupied droplet (with one biological particle) and a plurality of occupied droplets (with more than one biological particle). As described elsewhere herein, in some cases, the majority of occupied partitions may contain no more than one biological particle per occupied partition, and some of the generated partitions may be unoccupied (of any biological particle). However, in some cases, some of the occupied partitions may contain more than one biological particle. In some cases, the separation process may be controlled so that less than about 25% of the occupied partitions contain more than one biological particle, and in many cases, less than about 20% of the unoccupied partitions have more than one biological particle, and in some cases, less than about 10% or even less than about 5% of the occupied partitions contain more than one biological particle per partition.

[0079] In some cases, it may be desirable to minimize the creation of an excessive number of empty partitions, such as to reduce costs and / or improve efficiency. While such minimization may be achieved by providing a sufficient number of bioparticles (e.g., bioparticles 114) at separation junction 110, such as to ensure that at least one bioparticle is encapsulated in a partition, a Poissonian distribution may be expected to increase the number of partitions containing multiple bioparticles. Thus, in the case of obtaining a single occupied partition, a maximum of approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the generated partitions may be unoccupied.

[0080] In some cases, the flow of one or more biological particles (e.g., in channel segment 102) or other fluids directed to the separation junction (e.g., in channel segments 104, 106) can be controlled so that in many cases no more than about 50% of the generated partitions, no more than about 25% of the generated partitions, or no more than about 10% of the generated partitions are unoccupied. These flows can be controlled to present a non-Poisson distribution of a single occupied partition while providing a lower level of unoccupied partitions. The range of unoccupied partitions described above can be achieved while still providing any of the single occupancy rates described above. For example, in many cases, using the systems and methods described herein, an occupancy rate of less than about 25%, less than about 20%, less than about 15%, less than about 10%, and in many cases, less than about 5%, while having less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5% or less of the unoccupied partitions.

[0081] As will be appreciated, the occupancy described above also applies to partitions containing biological particles and additional agents, including but not limited to microcapsules carrying barcoded nucleic acid molecules (e.g., oligonucleotides) ( Figure 2 The occupied partitions (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the occupied partitions) can contain both microcapsules (e.g., beads) and bioparticles that include barcoded nucleic acid molecules.

[0082] On the other hand, in addition to or as an alternative to droplet-based separation, the bioparticles can be encapsulated in microcapsules, which include a shell, layer or porous matrix in which one or more individual bioparticles or small groups of bioparticles are entrained. The microcapsules may contain other agents. Encapsulation of bioparticles can be performed by a variety of processes. Such processes combine an aqueous fluid containing the bioparticles with a polymeric precursor material that can be capable of forming a gel or other solid or semi-solid matrix when a specific stimulus is applied to the polymer precursor. Such stimuli can include, for example, thermal stimuli (heating or cooling), light stimuli (e.g., by photocuring), chemical stimuli (e.g., by crosslinking, polymerization initiation of the precursor (e.g., by an added initiator), etc. and / or combinations of the above.

[0083] The preparation of microcapsules including bioparticles can be performed by a variety of methods. For example, an air knife droplet or aerosol generator can be used to dispense droplets of a precursor fluid into a gelling solution to form microcapsules containing a single bioparticle or small groups of bioparticles. Similarly, a membrane-based encapsulation system can be used to generate microcapsules including encapsulated bioparticles as described herein. The microfluidic system of the present disclosure, such as Figure 1The microfluidic system shown can be readily used to encapsulate cells as described herein. Specifically, and with reference to Figure 1 , an aqueous fluid 112 including (i) biological particles 114 and (ii) a polymer precursor material (not shown) flows into a channel junction 110, wherein the aqueous fluid is separated into droplets 118, 120 by the flow of a non-aqueous fluid 116. In the case of an encapsulation method, the non-aqueous fluid 116 may also include an initiator (not shown) to cause polymerization and / or cross-linking of the polymer precursor to form microcapsules containing the entrained biological particles. Examples of polymer precursor / initiator pairs include those described in U.S. Patent Application Publication No. 2014 / 0378345, which is incorporated herein by reference in its entirety for all purposes.

[0084] For example, where the polymer precursor material comprises a linear polymer material such as linear polyacrylamide, PEG or other linear polymeric material, the activator may comprise a crosslinker or a chemical that activates the crosslinker within the formed droplet. Similarly, for polymer precursors comprising polymerizable monomers, the activator may comprise a polymerization initiator. For example, in some cases, where the polymer precursor comprises a mixture of acrylamide monomers and N,N'-bis-(acryloyl)cystamine (BAC) comonomers, a reagent such as tetraethylmethylenediamine (TEMED) may be provided within the second fluid stream 116 in channel segments 104 and 106 that may initiate copolymerization of acrylamide and BAC into a crosslinked polymer network or hydrogel.

[0085] When the second fluid stream 116 contacts the first fluid stream 112 at the junction 110, during droplet formation, TEMED can diffuse from the second fluid 116 into the aqueous fluid 112 including the linear polyacrylamide, which will activate the crosslinking of the polyacrylamide within the droplets 118, 120, resulting in the formation of gel (e.g., hydrogel) microcapsules as solid or semi-solid beads or particles 114 entrapping cells. Although described in terms of polyacrylamide encapsulation, other "activatable" encapsulation compositions can also be employed in the context of the methods and compositions described herein. For example, alginate droplets are formed and then exposed to divalent metal ions (e.g., Ca 2+ Ions) can be used as an encapsulation process using the described process. Likewise, agarose droplets can also be converted into capsules by temperature-based gelation (e.g., cooling, etc.).

[0086] In some cases, the encapsulated bioparticles can be selectively released from the microcapsules, such as over time or upon application of a particular stimulus, sufficiently degrading the microcapsules to allow the bioparticles (e.g., cells) or their other contents to be released from the microcapsules, such as into partitions (e.g., droplets). For example, in the case of the polyacrylamide polymers described above, degradation of the microcapsules can be achieved by introducing a suitable reducing agent, such as DTT, etc., to cleave the disulfide bonds of the cross-linked polymer matrix. See, e.g., U.S. Patent Application Publication No. 2014 / 0378345, which is incorporated herein by reference in its entirety for all purposes.

[0087] Bioparticles can be subjected to other conditions sufficient to polymerize or gel the precursor. Conditions sufficient to polymerize or gel the precursor can include exposure to heating, cooling, electromagnetic radiation and / or light. Conditions sufficient to polymerize or gel the precursor can include any conditions sufficient to polymerize or gel the precursor. After polymerization or gelation, a polymer or gel can be formed around the bioparticle. The polymer or gel can be diffusion permeable for chemical or biochemical reagents. The polymer or gel can be diffusion impermeable for the macromolecular components of the bioparticle. In this way, the polymer or gel can work to allow the bioparticle to undergo chemical or biochemical operations while spatially limiting the macromolecular components to the droplet region defined by the polymer or gel. The polymer or gel can include disulfide cross-linked polyacrylamide, agarose, alginate, polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate, PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin or elastin. The polymer or gel may include any other polymer or gel.

[0088] Polymers or gels can be functionalized to bind targeted analytes such as nucleic acids, proteins, carbohydrates, lipids or other analytes. Polymers or gels can be polymerized or gelled by passive mechanisms. Polymers or gels can be stable under alkaline conditions or at elevated temperatures. Polymers or gels can have mechanical properties similar to the mechanical properties of beads. For example, polymers or gels can have a size similar to beads. Polymers or gels can have mechanical strengths (e.g., tensile strength) similar to the mechanical strengths of beads. The density of polymers or gels can be lower than that of oil. The density of polymers or gels can be roughly similar to that of buffers. Polymers or gels can have adjustable pore sizes. Apertures can be selected to, for example, retain denatured nucleic acids. Apertures can be selected to maintain diffusion permeability to exogenous chemicals such as sodium hydroxide (NaOH) and / or endogenous chemicals such as inhibitors. Polymers or gels can be biocompatible. Polymers or gels can maintain or enhance cell viability. Polymers or gels can be biochemically compatible. Polymers or gels can be polymerized and / or depolymerized in thermal, chemical, enzymatic and / or optical ways.

[0089] The polymer may include poly(acrylamide-co-acrylic acid) cross-linked with disulfide bonds. The preparation of the polymer may include a two-step reaction. In the first activation step, the poly(acrylamide-co-acrylic acid) may be exposed to an acylating agent to convert the carboxylic acid into an ester. For example, the poly(acrylamide-co-acrylic acid) may be exposed to 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM). The polyacrylamide-co-acrylic acid may be exposed to other salts of 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium. In the second cross-linking step, the ester formed in the first step may be exposed to a disulfide cross-linking agent. For example, the ester may be exposed to cystamine (2,2'-disulfide bis(ethylamine)). After these two steps, the bioparticles may be surrounded by polyacrylamide chains linked together by disulfide bonds. In this way, biological particles can be encapsulated inside a gel or matrix (e.g., a polymer matrix) or include a gel or matrix to form a "cell bead". The cell bead can contain biological particles (e.g., cells) or macromolecular components of biological particles (e.g., RNA, DNA, protein, etc.). The cell bead can contain a single cell or multiple cells or a derivative of a single cell or multiple cells. For example, after cell lysis and washing, the inhibitory components in the cell lysate can be washed away, and the macromolecular components can be combined into cell beads. The systems and methods disclosed herein can be applicable to both cell beads (and / or droplets or other partitions) containing biological particles and cell beads (and / or droplets or other partitions) containing macromolecular components of biological particles.

[0090] Encapsulated bioparticles can provide certain potential advantages of being easier to store and more portable than droplet-based separated bioparticles. In addition, in some cases, it may be necessary to incubate the bioparticles for a selected time period before analysis, such as to characterize the changes of such bioparticles over time in the presence or absence of different stimuli. In such cases, encapsulation can allow longer incubations than separation in emulsion droplets, but in some cases, droplet-separated bioparticles can also be incubated for different time periods, such as at least 10 seconds, at least 30 seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours or at least 10 hours or longer. The encapsulation of bioparticles can constitute the separation of bioparticles, and other reagents are separated into the bioparticles together. Alternatively or in addition, encapsulated bioparticles can be easily deposited into other partitions (e.g., droplets) as described above.

[0091] Partitions can include one or more unique identifiers such as barcodes. The barcode can be delivered to the partition containing compartmentalized or separated bioparticles before, after or simultaneously. For example, the barcode can be injected into the droplet before, after or simultaneously with the droplet generation. The delivery of the barcode to a specific partition allows the characteristics of the individual bioparticle to be attributed to the specific partition later. The barcode can be delivered to the partition by any suitable mechanism, for example, on a nucleic acid molecule (e.g., oligonucleotide). The barcoded nucleic acid molecule can be delivered to the partition by a microcapsule. In some cases, the barcoded nucleic acid molecule can be initially combined with the microcapsule, and then released from the microcapsule when a stimulus that allows the nucleic acid molecule to dissociate or release from the microcapsule is applied. In some cases, the microcapsule can include beads. Beads are described in further detail below.

[0092] Figure 2An example of a microfluidic channel structure 200 for delivering beads carrying barcodes to a droplet is shown. The channel structure 200 can include channel segments 201, 202, 204, 206, and 208 connected at a channel junction 210. In operation, the channel segment 201 can transport an aqueous fluid 212 containing a plurality of beads 214 (e.g., having nucleic acid molecules, oligonucleotides, molecular tags) along the channel segment 201 to the junction 210. The plurality of beads 214 can be derived from a suspension of beads. For example, the channel segment 201 can be connected to a reservoir comprising an aqueous suspension of beads 214. The channel segment 202 can transport an aqueous fluid 212 containing a plurality of biological particles 216 along the channel segment 202 to the junction 210. The plurality of biological particles 216 can be derived from a suspension of biological particles. For example, the channel segment 202 can be connected to a reservoir comprising an aqueous suspension of biological particles 216. In some cases, the aqueous fluid 212 in the first channel segment 201 or the second channel segment 202 or both segments can contain one or more reagents, as further described below. A second fluid 218 immiscible with the aqueous fluid 212 (e.g., oil) can be delivered to the junction 210 from each of the channel segments 204 and 206. When the aqueous fluid 212 from each of the channel segments 201 and 202 and the second fluid 218 from each of the channel segments 204 and 206 meet at the channel junction 210, the aqueous fluid 212 can be separated into discrete droplets 220 in the second fluid 218 and flow out of the junction 210 along the channel segment 208. The channel segment 208 can deliver the discrete droplets to an outlet reservoir fluidly coupled to the channel segment 208, where the discrete droplets can be harvested.

[0093] Alternatively, channel segments 201 and 202 may meet at another junction upstream of junction 210. At such a junction, the beads and bioparticles may form a mixture that is directed along another channel to junction 210 to produce droplets 220. The mixture may provide beads and bioparticles in an alternating manner, such that, for example, a droplet includes a single bead and a single bioparticle.

[0094] Beads, bioparticles and droplets can flow along the channel with a substantially regular flow distribution (e.g., with a regular flow rate). Such regular flow distribution can allow the droplets to contain a single bead and a single bioparticle. Such regular flow distribution can allow the occupancy of the droplets (e.g., droplets with beads and bioparticles) to be greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. For example, such regular flow distribution and devices that can be used to provide such regular flow distribution are provided in U.S. Patent Publication No. 2015 / 0292988, which is incorporated herein by reference in its entirety.

[0095] The second fluid 218 may include an oil, such as a fluorinated oil, that contains a fluorinated surfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets 220 .

[0096] The generated discrete droplets may contain a single biological particle 216. The generated discrete droplets may contain a bead 214 carrying a barcode or other reagent. The generated discrete droplets may contain both a single biological particle and a bead carrying a barcode, such as droplet 220. In some cases, the discrete droplets may contain more than one single biological particle or no biological particles. In some cases, the discrete droplets may contain more than one bead or no beads. The discrete droplets may be unoccupied (e.g., no beads, no biological particles).

[0097] Advantageously, separating discrete droplets of bioparticles and beads carrying barcodes can effectively allow attribution of barcodes to macromolecular components of the bioparticles within a partition.The contents of a partition can be kept separate from the contents of other partitions.

[0098] It should be understood that the channel section described herein can be coupled to any one of a variety of different fluid sources or receiving components, including fluid components of reservoirs, pipes, manifolds or other systems.It should be understood that microfluidic channel structure 200 can have other geometric shapes.For example, the microfluidic channel structure can have more than one channel junction.For example, the microfluidic channel structure can have 2, 3, 4 or 5 channel sections, and each channel section carries the beads met at the channel junction.Fluid can flow along one or more channels or reservoirs via one or more fluid flow units.Fluid flow units can include compressors (for example, positive pressure is provided), pumps (for example, negative pressure is provided), actuators, etc. to control the flow of fluid.Fluid can also or otherwise be controlled by applied pressure differentials, centrifugal forces, electric pumps, vacuum, capillaries or gravity flows, etc.

[0099] The beads can be porous, non-porous, solid, semi-solid, semi-fluid, fluid and / or combinations thereof. In some cases, the beads can be soluble, destructible and / or degradable. In some cases, the beads can be non-degradable. In some cases, the beads can be gel beads. The gel beads can be hydrogel beads. The gel beads can be formed by molecular precursors such as polymeric or monomeric species. Semi-solid beads can be liposome beads. Solid beads can include metals, the metals comprising iron oxide, gold and silver. In some cases, beads can be silica beads. In some cases, beads can be rigid. In other cases, beads can be flexible and / or compressible.

[0100] The beads can be any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non-spherical, elliptical, rectangular, amorphous, circular, cylindrical, and variations thereof.

[0101] The beads can be of uniform size or of uneven size. In some cases, the diameter of the beads can be at least about 1 micrometer (μm), 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1 mm or more. In some cases, the diameter of the beads may be less than about 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1 mm or less. In some cases, the diameter of the beads may be in the range of about 40-75 μm, 30-75 μm, 20-75 μm, 40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250 μm, or 20-500 μm.

[0102] In some aspects, beads can be provided as a population or a plurality of beads having a relatively monodisperse size distribution. Where it may be desirable to provide a relatively consistent amount of reagent within a partition, maintaining relatively consistent bead properties such as size can contribute to overall consistency. Specifically, beads described herein can have a size distribution having a coefficient of variation of its cross-sectional dimensions of less than 50%, less than 40%, less than 30%, less than 20%, and in some cases less than 15%, less than 10%, less than 5%, or less.

[0103] Beads can include natural and / or synthetic materials. For example, beads can include natural polymers, synthetic polymers, or both natural and synthetic polymers. Examples of natural polymers include proteins and sugars such as deoxyribonucleic acid, rubber, cellulose, starch (e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silk, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan, psyllium ovata, gum arabic, agar, gelatin, shellac, karaya gum, xanthan gum, corn gum, guar gum, karaya gum, agarose, alginic acid, alginate, or its natural polymers. Examples of synthetic polymers include acrylic, nylon, silicone, spandex, viscose rayon, polycarboxylic acid, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol, polyurethane, polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene, polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(ethylene terephthalate), polyethylene, polyisobutylene, poly(methyl methacrylate), poly(formaldehyde), polyoxymethylene, polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene chloride), poly(vinylidene fluoride), poly(vinyl fluoride), and / or combinations (e.g., copolymers) thereof. Beads may also be formed from materials other than polymers, including lipids, micelles, ceramics, glass ceramics, material composites, metals, other inorganic materials, and the like.

[0104] In some cases, beads can contain molecular precursors (e.g., monomers or polymers), which can form polymer networks by the polymerization of molecular precursors. In some cases, precursors can be species that have been polymerized that can be further polymerized by, for example, chemical crosslinking. In some cases, precursors can include one or more of acrylamide or methacrylamide monomers, oligomers or polymers. In some cases, beads can include prepolymers, which are oligomers that can be further polymerized. For example, polyurethane beads can be prepared using prepolymers. In some cases, beads can contain single polymers that can be further polymerized. In some cases, beads can be generated by the polymerization of different precursors so that they include mixed polymers, copolymers and / or block copolymers. In some cases, beads can include covalent bonds or ionic bonds between polymerization precursors (e.g., monomers, oligomers, linear polymers), nucleic acid molecules (e.g., oligonucleotides), primers and other entities. In some cases, covalent bonds can be carbon-carbon bonds or thioether bonds.

[0105] Crosslinking can be permanent or reversible, depending on the specific crosslinking agent used. Reversible crosslinking can allow the polymer to linearize or dissociate under appropriate conditions. In some cases, reversible crosslinking can also allow materials bound to the bead surface to be reversibly connected. In some cases, the crosslinking agent can form disulfide bonds. In some cases, the chemical crosslinking agent that forms disulfide bonds can be cystamine or modified cystamine.

[0106] In some cases, disulfide bonds can be formed between molecular precursor units (e.g., monomers, oligomers, or linear polymers) or precursors incorporated into beads and nucleic acid molecules (e.g., oligonucleotides). For example, cystamine (including modified cystamine) is an organic reagent that includes disulfide bonds that can be used as a cross-linker between individual monomers or polymeric precursors of beads. Polyacrylamide can be polymerized in the presence of cystamine or a species that includes cystamine (e.g., modified cystamine) to generate polyacrylamide gel beads that include disulfide bonds (e.g., chemically degradable beads that include a chemically reducing cross-linker). The disulfide bonds can allow the beads to degrade (or dissolve) when the beads are exposed to a reducing agent.

[0107] In some cases, chitosan, a linear polysaccharide polymer, can be cross-linked with glutaraldehyde via hydrophilic chains to form beads. Cross-linking of chitosan polymers can be achieved by chemical reactions initiated by heat, pressure, pH changes, and / or radiation.

[0108] In some cases, beads can include an acrylamide moiety that can be used to connect one or more nucleic acid molecules (e.g., barcode sequences, barcoded nucleic acid molecules, barcoded oligonucleotides, primers, or other oligonucleotides) to beads in some aspects. In some cases, the acrylamide moiety can refer to an acrylamide analog generated by the reaction of acrylamide with one or more species, such as the reaction of acrylamide with other monomers and crosslinking agents during polymerization. The acrylamide moiety can be modified to form a chemical bond with the species to be connected, such as a nucleic acid molecule (e.g., barcode sequence, barcoded nucleic acid molecule, barcoded oligonucleotide, primers, or other oligonucleotides). The acrylamide moiety can be modified with a thiol group that can form a disulfide bond or can be modified with a group that already includes a disulfide bond. Thiol or disulfide (via disulfide exchange) can be used as an anchor point for the species to be connected or another portion of the acrylamide moiety can be used for connection. In some cases, the linkage may be reversible, such that when the disulfide bond is broken (eg, in the presence of a reducing agent), the linked species is released from the bead. In other cases, the acrylamido moiety may include a reactive hydroxyl group that can be used for linkage.

[0109] Functionalization of beads for attachment of nucleic acid molecules (e.g., oligonucleotides) can be achieved by a variety of different methods, including activation of chemical groups within the polymer, incorporation of active or activatable functional groups into the polymer structure, or attachment at the prepolymer or monomer stage in bead generation.

[0110] For example, the precursors (e.g., monomers, cross-linking agents) that are polymerized to form beads can include an acrylamide moiety such that when beads are generated, the beads also include an acrylamide moiety. The acrylamide moiety can be attached to a nucleic acid molecule (e.g., an oligonucleotide), which can include a priming sequence (e.g., a primer for amplifying a target nucleic acid, a random primer, a primer sequence for messenger RNA) and / or one or more barcode sequences. The one or more barcode sequences can include the same sequence as all nucleic acid molecules coupled to a given bead and / or a sequence different from all nucleic acid molecules coupled to a given bead. Nucleic acid molecules can be incorporated into beads.

[0111] In some cases, the nucleic acid molecule may include a functional sequence, such as for attachment to a sequencing flow cell, such as for In some cases, the nucleic acid molecule may include a barcode sequence. In some cases, the primer may further include a unique molecular identifier (UMI). In some cases, the primer may include an R1 primer sequence for Illumina sequencing. In some cases, the primer may include an R2 primer sequence for Illumina sequencing. Examples of such nucleic acid molecules (e.g., oligonucleotides, polynucleotides, etc.) and their uses that can be used with the compositions, devices, methods and systems of the present disclosure are provided in U.S. Patent Publication Nos. 2014 / 0378345 and 2015 / 0376609, each of which is incorporated herein by reference in its entirety.

[0112] In some cases, precursors that include reactive or activated functional groups to make them reactive can be polymerized with other precursors to generate gel beads that include activated or activatable functional groups. Functional groups can then be used to connect other species (e.g., disulfide linkers, primers, other oligonucleotides, etc.) to the gel beads. For example, some precursors that include carboxylic acid (COOH) groups can be copolymerized with other precursors to form gel beads that also include COOH functional groups. In some cases, acrylic acid (a species that includes free COOH groups), acrylamide, and bis(acryloyl)cystamine can be copolymerized together to generate gel beads that include free COOH groups. The COOH groups of the gel beads can be activated (e.g., by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)) to render the groups reactive (e.g., to react with amine functional groups in the case where EDC / NHS or DMTMM is used for activation). The activated COOH groups can then be reacted with a suitable species (e.g., a species including an amine functional group in which the carboxylic acid group is activated to react with the amine functional group) that includes the moiety to be attached to the beads.

[0113] Beads including disulfide bonds in their polymeric network can be functionalized with additional species by reducing some of the disulfide bonds to free thiols. Disulfide bonds can be reduced to generate free thiol groups by the action of, for example, a reducing agent (e.g., DTT, TCEP, etc.) without dissolving the beads. The free thiols of the beads can then react with the free thiols of the species or species including another disulfide bond (e.g., by thiol-disulfide exchange) so that the species can be connected to the beads (e.g., by the generated disulfide bonds). In some cases, the free thiols of the beads can react with any other suitable groups. For example, the free thiols of the beads can react with species including acrylamide moieties. The free thiol groups of the beads can react with acrylamide groups by Michael addition chemistry so that species including acrylamide groups are connected to the beads. In some cases, uncontrolled reactions can be prevented by including thiol capping agents such as N-ethylmaleimide or iodoacetate.

[0114] The activation of disulfide bonds within the beads can be controlled so that only a small number of disulfide bonds are activated. For example, this can be controlled by controlling the concentration of the reducing agent used to generate free thiol groups and / or the concentration of the reagent used to form disulfide bonds in the bead polymerization. In some cases, a low concentration (e.g., a reducing agent molecule: gel bead ratio of less than or equal to about 1:100,000,000,000, less than or equal to about 1:10,000,000,000, less than or equal to about 1:1,000,000,000, less than or equal to about 1:100,000,000, less than or equal to about 1:10,000,000, less than or equal to about 1:1,000,000,000, less than or equal to about 1:100,000,000, less than or equal to about 1:1,000,000, less than or equal to about 1:100,000, less than or equal to about 1:10,000) of the reducing agent can be used for reduction. Controlling the number of disulfide bonds reduced to free thiols can help ensure the structural integrity of the beads during functionalization. In some cases, optically active agents such as fluorescent dyes can be coupled to the beads via their free thiol groups and used to quantify the number of free thiols present in the beads and / or to track the beads.

[0115] In some cases, it can be advantageous to add a part to the gel beads after the gel beads are formed. For example, adding oligonucleotides (for example, barcoded oligonucleotides) after the gel beads are formed can avoid the loss of species during the chain transfer termination period that may occur during polymerization. In addition, smaller precursors (for example, monomers or cross-linking agents that do not include side chain groups and connecting parts) can be used for polymerization and can hinder the chain ends of growth to a minimum due to the viscosity effect. In some cases, the functionalization after the gel beads are synthesized can minimize the exposure of species (for example, oligonucleotides) loaded with potential destructive agents (for example, free radicals) and / or chemical environments. In some cases, the gel generated can have an upper critical solution temperature (UCST), and the temperature allows the beads to expand and collapse due to temperature. During the subsequent functionalization of the beads with oligonucleotides, such functionality can contribute to the oligonucleotides (for example, primers) to penetrate into the beads. Functionalization after production can also be used to control the loading rate of species in the beads, so that, for example, the variability of the loading rate is minimized. Species loading can also be performed in a batch process, so that multiple beads can be functionalized with species in a single batch.

[0116] The beads injected or otherwise introduced into the partitions may include a releasable, cleavable or reversibly attached barcode. The beads injected or otherwise introduced into the partitions may include an activatable barcode. The beads injected or otherwise introduced into the partitions may be degradable, destructible or soluble beads.

[0117] The barcode can be releasable, cleavable or reversibly connected to the bead, so that the barcode can be released by cutting the bond between the barcode molecule and the bead or can be released or released by the degradation of the underlying bead itself, thereby allowing access to the barcode or being accessible to other reagents or both. In a non-limiting example, the cutting can be achieved by reducing disulfide bonds, using restriction enzymes, photoactivated cutting or by cutting and / or reaction of other types of stimulation (e.g., chemical, thermal, pH, enzymatic, etc.), as described elsewhere herein. Releasable barcodes can sometimes be referred to as activatable because once released, the barcode can be used for reaction. Therefore, for example, an activatable barcode can be activated by releasing the barcode from the bead (or other suitable types of partitions described herein). Other activatable configurations are also contemplated in the context of the described methods and systems.

[0118] In addition to or as an alternative to the cleavable bonds between beads and related molecules such as nucleic acid molecules containing barcodes (e.g., barcoded oligonucleotides), beads can be degradable, rupturable, or soluble spontaneously or when exposed to one or more stimuli (e.g., temperature changes, pH changes, exposure to specific chemical species or phases, exposure to light, reducing agents, etc.). In some cases, beads can be soluble so that the material components of the beads dissolve when exposed to specific chemical species or environmental changes such as temperature changes or pH changes. In some cases, gel beads can degrade or dissolve under high temperature and / or alkaline conditions. In some cases, beads can be thermally degradable so that when the beads are exposed to appropriate temperature changes (e.g., heat), the beads degrade. Degradation or dissolution of beads bound to species (e.g., nucleic acid molecules, e.g., barcoded oligonucleotides) may result in the release of species from the beads.

[0119] It should be understood from the above disclosure that degradation of beads can refer to the dissociation of bound or entrained species from beads with and without structural degradation of the physical beads themselves. For example, degradation of beads can involve cutting cleavable bonds by one or more species and / or methods described elsewhere herein. In another example, entrained species can be released from beads by osmotic pressure differences, for example, due to changes in the chemical environment. For example, changes in the pore size of beads caused by osmotic pressure differences can generally occur without structural degradation of the beads themselves. In some cases, the increase in pore size due to osmotic swelling of beads can allow entrained species to be released within the beads. In other cases, due to pore size shrinkage, osmotic shrinkage of beads can enable beads to better retain entrained species.

[0120] Degradable beads can be introduced into partitions such as droplets or wells of an emulsion so that the beads degrade within the partitions and any related species (e.g., oligonucleotides) are released within the droplets when appropriate stimuli are applied. Free species (e.g., oligonucleotides, nucleic acid molecules) can interact with other reagents contained in the partitions. For example, polyacrylamide beads including cystamine and connected to a barcode sequence via a disulfide bond can be combined with a reducing agent within an oil-in-water emulsion droplet. Within the droplet, the reducing agent can break various disulfide bonds, causing the beads to degrade and release the barcode sequence into the aqueous internal environment of the droplet. In another example, heating a droplet including a bead-bound barcode sequence in an alkaline solution may also result in bead degradation and release of the connected barcode sequence into the aqueous internal environment of the droplet.

[0121] Any suitable number of molecular tag molecules (e.g., primers, barcoded oligonucleotides) can be associated with beads so that upon release from the beads, the molecular tag molecules (e.g., primers, e.g., barcoded oligonucleotides) are present in the partitions at predefined concentrations. Such predefined concentrations can be selected to facilitate certain reactions, such as amplification, for generating sequencing libraries within the partitions. In some cases, the predefined concentrations of primers can be limited by the process of generating beads with nucleic acid molecules (e.g., oligonucleotides).

[0122] In some cases, beads can be non-covalently loaded with one or more reagents. Beads can be non-covalently loaded by, for example, subjecting beads to conditions sufficient to swell the beads, allowing reagents to have enough time to diffuse into the inside of the beads, and subjecting beads to conditions sufficient to deswell the beads. The swelling of beads can be achieved, for example, by placing the beads in a thermodynamically favorable solvent, subjecting the beads to a higher or lower temperature, subjecting the beads to a higher or lower ion concentration, and / or subjecting the beads to an electric field. The swelling of beads can be achieved by various swelling methods. The deswelling of beads can be achieved by, for example, transferring the beads to a thermodynamically unfavorable solvent, subjecting the beads to a lower or high temperature, subjecting the beads to a lower or higher ion concentration, and / or removing the electric field. The deswelling of beads can be achieved by various deswelling methods. Transferring beads may cause the holes in the beads to shrink. Then, shrinkage can hinder the reagent in the beads from diffusing out from the inside of the beads. The hindrance may be due to the spatial interaction between the reagent and the inside of the beads. Transfer can be achieved in a microfluidic manner. For example, the transfer can be achieved by moving the beads from one co-flowing solvent stream to a different co-flowing solvent stream.The swelling properties and / or pore size of the beads can be adjusted by varying the polymer composition of the beads.

[0123] In some cases, the acrylamide moiety attached to the precursor, another species attached to the precursor, or the precursor itself can include labile bonds such as chemical, heat or light sensitive bonds, such as disulfide bonds, UV sensitive bonds, etc. Once the acrylamide moiety or other moiety including an unstable bond is incorporated into the bead, the bead can also include an unstable bond. For example, an unstable bond can be used to reversibly link (e.g., covalently link) a species (e.g., a barcode, a primer, etc.) to the bead. In some cases, a thermally labile bond can include a connection based on nucleic acid hybridization, for example, where an oligonucleotide hybridizes to a complementary sequence attached to the bead, such that thermal melting of the hybrid releases an oligonucleotide from the bead or microcapsule, such as a sequence containing a barcode.

[0124] Adding multiple types of unstable bonds to gel beads may produce beads that can respond to various stimuli. Each type of unstable bond may be sensitive to associated stimuli (e.g., chemical stimuli, light, temperature, enzymatic, etc.), so that the release of species connected to the beads by each unstable bond can be controlled by applying appropriate stimuli. Such functionality can be used to control the release of species from gel beads. In some cases, after the gel beads are formed, another species including an unstable bond can be connected to the gel beads by, for example, an activated functional group of the gel beads described above. It should be understood that the barcodes that can be releasably, cleavably or reversibly connected to the beads described herein include barcodes that are released by cutting the bond between the barcode molecule and the beads or that can be released or released by degradation of the underlying beads themselves, thereby allowing access to the barcode or being accessible to other reagents or both.

[0125] Releasable barcodes as described herein may sometimes be referred to as activatable because once released, the barcodes are available for reaction. Thus, for example, an activatable barcode may be activated by releasing the barcode from a bead (or other suitable type of partition described herein). Other activatable configurations are also contemplated in the context of the described methods and systems.

[0126] In addition to thermally cleavable bonds, disulfide bonds, and UV-sensitive bonds, other non-limiting examples of labile bonds that can be coupled to the precursor or bead include ester bonds (e.g., cleavable by acid, base, or hydroxylamine), carbamate bonds (e.g., cleavable by diethylenetriamine "DETA"), vicinal diol bonds (e.g., cleavable by sodium periodate), Diels-Alder bonds (e.g., cleavable by heat), sulfone bonds (e.g., cleavable by base), silyl ether bonds (e.g., cleavable by acid), glycosidic bonds (e.g., cleavable by amylases), peptide bonds (e.g., cleavable by proteases), or phosphodiester bonds (e.g., cleavable by nucleases (e.g., DNases)). As further described below, bonds can be cleaved by other nucleic acid molecules that target enzymes such as restriction enzymes (e.g., restriction endonucleases).

[0127] During bead generation (e.g., during polymerization of a precursor), species can be encapsulated in the beads. Such species may or may not participate in polymerization. Such species can be added to a polymerization reaction mixture so that the resulting beads include the species when the beads are formed. In some cases, such species can be added to the gel beads after formation. Such species can include, for example, nucleic acid molecules (e.g., oligonucleotides), reagents for nucleic acid amplification reactions including those described herein (e.g., primers, polymerases, dNTPs, cofactors (e.g., ionic cofactors), buffers), reagents for enzymatic reactions (e.g., enzymes, cofactors, substrates, buffers), reagents for nucleic acid modification reactions such as polymerization, ligation or digestion, and / or reagents for one or more sequencing platforms (e.g., for of ) template preparation (e.g., labeling) reagents. Such species may include one or more enzymes described herein, including but not limited to polymerases, reverse transcriptases, restriction enzymes (e.g., endonucleases), transposases, ligases, proteinase K, DNases, etc. Such species may include one or more reagents described elsewhere herein (e.g., cleavage agents, inhibitors, inactivators, chelators, stimulation). The capture of such species may be controlled by the density of the polymer network generated during the polymerization of the precursor, the control of the ionic charge in the gel beads (e.g., by ionic species connected to polymer species), or by the release of other species. When the beads degrade and / or by applying a stimulus that can release the species from the beads, the encapsulated species can be released from the beads. Alternatively or in addition, during or after the partition formation, the species can be separated in the partition (e.g., droplets). Such species may include but are not limited to the above-mentioned species that can also be encapsulated in the beads.

[0128] Degradable beads can include one or more species with unstable bonds, so that when the beads / species are exposed to an appropriate stimulus, the bonds break and the beads degrade. The unstable bonds can be chemical bonds (e.g., covalent bonds, ionic bonds) or can be another type of physical interaction (e.g., van der Waals interactions, dipole-dipole interactions, etc.). In some cases, the cross-linking agent used to generate the beads can include unstable bonds. When exposed to appropriate conditions, the unstable bonds can break and the beads degrade. For example, when polyacrylamide gel beads including cystamine cross-linkers are exposed to a reducing agent, the disulfide bonds of cystamine can break and the beads degrade.

[0129] Compared with non-degradable beads, when appropriate stimulation is applied to the beads, degradable beads can be used to release the species (e.g., nucleic acid molecules, barcode sequences, primers, etc.) connected from the beads more quickly. For example, for species bound to the inner surface of porous beads or in the case of encapsulated species, the species can have greater mobility and accessibility to other species in solution when the beads are degraded. In some cases, the species can also be connected to the degradable beads by a degradable linker (e.g., a disulfide linker). The degradable linker can react to the same stimulation as the degradable beads, or two degradable species can react to different stimulations. For example, the barcode sequence can be connected to the polyacrylamide beads including cystamine by a disulfide bond. When the barcoded beads are exposed to a reducing agent, the beads are degraded, and the barcode sequence is released when the disulfide bond between the barcode sequence and the beads and the disulfide bond of cystamine in the beads are broken.

[0130] It should be understood from the above disclosure that, although referred to as degradation of the beads in many cases as described above, the degradation can refer to the dissociation of bound or entrapped species from the beads with and without structurally degrading the physical beads themselves. For example, entrapped species can be released from the beads by osmotic pressure differentials, such as due to changes in the chemical environment. For example, changes in the pore size of the beads due to osmotic pressure differentials can generally occur without structural degradation of the beads themselves. In some cases, the increase in pore size due to osmotic swelling of the beads can allow the release of entrapped species within the beads. In other cases, osmotic shrinkage of the beads can allow the beads to better retain entrapped species due to shrinkage of the pore size.

[0131] In the case of providing degradable beads, it may be beneficial to avoid exposing such beads to one or more stimuli that cause such degradation before a given time, so as to avoid premature bead degradation and problems caused by such degradation, including, for example, poor flow characteristics and aggregation. For example, in the case where the beads include reducible cross-linking groups such as disulfide groups, it is desirable to avoid contacting such beads with reducing agents, such as DTT or other disulfide bond cleavage agents. In such cases, the treatment of the beads described herein will not provide reducing agents such as DTT in some cases. Because reducing agents are often provided in commercial enzyme preparations, it is desirable to provide an enzyme preparation that does not contain a reducing agent (or does not contain DTT) when treating the beads described herein. Examples of such enzymes include, for example, polymerase preparations, reverse transcriptase preparations, ligase preparations, and many other enzyme preparations that can be used to treat the beads described herein. The term "reducing agent-free" or "DTT-free" preparation may refer to a preparation having a lower limit range of less than about 1 / 10, less than about 1 / 50, or even less than about 1 / 100 of such materials for degrading beads. For example, for DTT, a formulation without a reducing agent may have less than about 0.01 millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even less than about 0.0001 mM DTT. In many cases, the amount of DTT may be undetectable.

[0132] Many chemical triggers can be used to trigger the degradation of beads. Examples of these chemical changes can include, but are not limited to, pH-mediated changes in the integrity of components within the beads, degradation of bead components through cleavage of cross-links, and depolymerization of bead components.

[0133] In some embodiments, beads can be formed by materials including degradable chemical crosslinkers such as BAC or cystamine. The degradation of such degradable crosslinkers can be achieved by a variety of mechanisms. In some instances, beads can be contacted with chemical degradation agents, which can cause oxidation, reduction or other chemical changes. For example, chemical degradation agents can be reducing agents such as dithiothreitol (DTT). Other examples of reducing agents can include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris (2-carboxyethyl) phosphine (TCEP) or a combination thereof. The reducing agent can degrade the disulfide bonds formed between the gel precursors that form beads, thereby degrading the beads. In other cases, changes in the pH of the solution such as an increase in pH may trigger the degradation of beads. In other cases, exposure to aqueous solutions such as water may trigger hydrolytic degradation, thereby causing degradation of beads.

[0134] The beads can also be induced to release their contents upon application of a thermal stimulus. Changes in temperature can cause various changes in the beads. For example, heat can cause solid beads to liquefy. Changes in heat can cause the beads to melt, causing a portion of the beads to degrade. In other cases, heat can increase the internal pressure of the bead components, causing the beads to rupture or explode. Heat can also act on heat-sensitive polymers used as materials for building beads.

[0135] Any suitable agent can degrade beads. In certain embodiments, the change of temperature or pH can be used to degrade the heat-sensitive or pH-sensitive bonds in the beads. In certain embodiments, chemical degradation agents can be used to degrade the chemical bonds in the beads by oxidation, reduction or other chemical changes. For example, chemical degradation agents can be reducing agents such as DTT, wherein DTT can degrade the disulfide bonds formed between the cross-linking agent and the gel precursor, thereby degrading the beads. In certain embodiments, reducing agents can be added to degrade the beads, which may or may not cause the beads to release their contents. Examples of reducing agents can include dithiothreitol (DTT), β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris (2-carboxyethyl) phosphine (TCEP) or a combination thereof. Reducing agents can be present at a concentration of about 0.1mM, 0.5mM, 1mM, 5mM, 10mM. The reducing agent may be present at a concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, or greater than 10 mM. The reducing agent may be present at a concentration of up to about 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM, or less.

[0136] Any suitable number of molecular tag molecules (e.g., primers, barcoded oligonucleotides) can be associated with beads so that upon release from the beads, the molecular tag molecules (e.g., primers, e.g., barcoded oligonucleotides) are present in the partitions at predefined concentrations. Such predefined concentrations can be selected to facilitate certain reactions, such as amplification, for generating sequencing libraries within the partitions. In some cases, the predefined concentrations of primers can be limited by the process of generating beads with oligonucleotides.

[0137] Although Figure 1 and Figure 2It has been described above in terms of providing a substantially single occupied partition, but in some cases it may be desirable to provide a plurality of occupied partitions, e.g., containing two, three, four or more cells and / or microcapsules (e.g., beads) comprising a barcoded nucleic acid molecule (e.g., oligonucleotide) within a single partition. Thus, as described above, the flow characteristics of the bioparticles and / or beads containing the fluid and the separation fluid can be controlled to provide such a plurality of occupied partitions. Specifically, the flow parameters can be controlled to provide a given occupancy of greater than about 50% of a partition, greater than about 75%, and in some cases greater than about 80%, 90%, 95%, or more.

[0138] In some cases, additional microcapsules may be used to deliver additional reagents to the partitions. In such cases, it may be advantageous to introduce different beads into a common channel or droplet generation junction from different sources of beads (e.g., containing different associated reagents) through different channel inlets into such a common channel or droplet generation junction (e.g., junction 210). In such cases, the flow and frequency of different beads entering the channel or junction may be controlled to provide a specific ratio of microcapsules from each source while ensuring a given pairing or combination of such beads into a partition with a given number of bioparticles (e.g., one bioparticle and one bead per partition).

[0139] The partitions described herein can include small volumes, such as less than about 10 microliters (μL), 5 μL, 1 μL, 900 picoliters (pL), 800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

[0140] For example, in the case of droplet-based partitioning, the total volume of the droplets can be less than about 1000 pL, 900 pL, 800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10 pL, 1 pL or less. In the case of co-isolation with microcapsules, it should be understood that the volume of sample fluid within the partition, e.g., containing co-isolated bioparticles and / or beads, can be less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the volumes described above.

[0141] As described in other places of this paper, separation species can generate partition colony or multiple partitions.In such cases, the partition of any suitable quantity can be generated or otherwise provided.For example, at least about 1,000 partitions, at least about 5,000 partitions, at least about 10,000 partitions, at least about 50,000 partitions, at least about 100,000 partitions, at least about 500,000 partitions, at least about 1,000,000 partitions, at least about 5,000,000 partitions, at least about 10,000,000 partitions, at least about 50,000,000 partitions, at least about 100,000,000 partitions, at least about 500,000,000 partitions, at least about 100,000,000 partitions, at least about 500,000,000 partitions, at least about 1,000,000,000 partitions or more partitions can be generated or otherwise provided.In addition, described multiple partitions can comprise unoccupied partitions (for example, empty partitions) and the partitions occupied.

[0142] According to certain aspects, the biological particles can be separated together with a lysis reagent to release the contents of the biological particles within the partition. In such cases, the lysis reagent can be contacted with the biological particle suspension at the same time as or immediately before the biological particles are introduced into the separation junction / droplet generation area (e.g., junction 210), such as through one or more additional channels upstream of the channel junction. According to other aspects, additionally or alternatively, the biological particles can be separated together with other reagents, as will be further described below.

[0143] Figure 3 An example of a microfluidic channel structure 300 for co-separating biological particles and reagents is shown. Channel structure 300 may include channel segments 301, 302, 304, 306, and 308. Channel segments 301 and 302 are connected at a first channel junction 309. Channel segments 302, 304, 306, and 308 are connected at a second channel junction 310.

[0144] In an example operation, channel segment 301 can transport aqueous fluid 312 containing a plurality of biological particles 314 along channel segment 301 into second junction 310. Alternatively or additionally, channel segment 301 can transport beads (eg, gel beads). The beads can include barcode molecules.

[0145] For example, channel segment 301 can be connected to a reservoir of an aqueous suspension including biological particles 314. Upstream of second junction 310 and immediately before reaching it, channel segment 301 can meet channel segment 302 at first junction 309. Channel segment 302 can transport multiple reagents 315 (e.g., lysing agents) suspended in aqueous fluid 312 along channel segment 302 into first junction 309. For example, channel segment 302 can be connected to a reservoir including reagents 315. After first junction 309, aqueous fluid 312 in channel segment 301 can carry both biological particles 314 and reagents 315 toward second junction 310. In some cases, aqueous fluid 312 in channel segment 301 can contain one or more reagents, which can be the same or different reagents as reagent 315. A second fluid 316 immiscible with aqueous fluid 312 (e.g., oil) can be delivered to second junction 310 from each of channel segments 304 and 306. When the aqueous fluid 312 from channel segment 301 and the second fluid 316 from each of channel segments 304 and 306 meet at the second channel junction 310, the aqueous fluid 312 can be separated into discrete droplets 318 in the second fluid 316 and flow out of the second junction 310 along the channel segment 308. The channel segment 308 can deliver the discrete droplets 318 to an outlet reservoir fluidly coupled to the channel segment 308, where the discrete droplets can be harvested.

[0146] The second fluid 316 may include an oil, such as a fluorinated oil, that contains a fluorinated surfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets 318 .

[0147] The generated discrete droplets may contain a single biological particle 314 and / or one or more reagents 315. In some cases, the generated discrete droplets may contain beads (not shown) carrying a barcode, such as through other microfluidic structures described elsewhere herein. In some cases, the discrete droplets may be unoccupied (e.g., no reagents, no biological particles).

[0148] Advantageously, when the lysis reagent and the biological particles are separated together, the lysis reagent can promote the release of the contents of the biological particles in the partition. The contents released in the partition can be kept separate from the contents of other partitions.

[0149] It should be understood that the passage section described herein can be coupled to any one of a variety of different fluid sources or receiving components, including fluid components of reservoirs, pipes, manifolds or other systems.It should be understood that the microfluidic channel structure 300 can have other geometric shapes.For example, the microfluidic channel structure can have more than two channel junctions.For example, the microfluidic channel structure can have 2, 3, 4, 5 or more passage sections, and each passage section carries the same or different types of beads, reagents and / or bioparticles that meet at the passage junction.The fluid flow in each passage section can be controlled to control the separation of different elements into droplets.Fluid can flow along one or more channels or reservoirs via one or more fluid flow units.The fluid flow unit can include a compressor (for example, positive pressure is provided), a pump (for example, negative pressure is provided), an actuator, etc. to control the flow of fluid.Fluid can also or otherwise be controlled by the pressure difference, centrifugal force, electric pump, vacuum, capillary or gravity flow, etc. applied.

[0150] The example of lysing agent comprises bioactive reagent, such as lysing enzyme for cracking different cell types such as Gram-positive or negative bacteria, plants, yeast, mammals, etc., such as lysozyme, colorless peptidase, lysostaphin, labiase, cell lysing enzyme, lysing enzyme and multiple can be obtained from other lysing enzymes such as Sigma-Aldrich, Inc. (St. Louis, Missouri) and other commercially available lysing enzymes. Other lysing agents can be separated from biological particles in addition or alternatively to cause the contents of biological particles to be released into the partition. For example, in some cases, the lysing solution based on surfactant can be used to crack cells, but these may be less ideal for the system based on emulsion that surfactant can interfere with stable emulsion. In some cases, the lysing solution can include nonionic surfactants, such as TritonX-100 and Tween20. In some cases, the lysing solution can include ionic surfactants, such as sodium lauryl sarcosine and sodium lauryl sulfate (SDS). Electroporation, thermal, acoustic, or mechanical cell disruption may also be used in some cases, for example, non-emulsion-based separations such as encapsulation of bioparticles may supplement or replace droplet separations, where any pore size of the encapsulation is small enough to retain nucleic acid fragments of a given size after cell disruption.

[0151] In addition to the lysing agent co-separated with the bioparticles described above, other reagents can also be co-separated with the bioparticles, including for example DNase and RNase inactivators or inhibitors, such as proteinase K, chelating agents, such as EDTA and other reagents for removing or otherwise reducing negative activity or the impact of different cell lysate components on nucleic acid subsequent treatment. In addition, in the case of encapsulated bioparticles, the bioparticles can be exposed to appropriate stimulation to release the bioparticles or their content from the co-separated microcapsules. For example, in some cases, chemical stimulation can be co-separated with the encapsulated bioparticles to allow microcapsules to degrade and release cells or their content into larger partitions. In some cases, this stimulation can be the same as the stimulation described elsewhere herein for nucleic acid molecules (for example, oligonucleotides) released from their corresponding microcapsules (for example, beads). In alternative terms, this can be different and non-overlapping stimulations to allow the encapsulated bioparticles to be released into the partitions at a time different from the nucleic acid molecules being released into the same partition.

[0152] Additional reagents may also be co-separated with the bioparticles, such as endonucleases, DNA polymerases, and dNTPs for amplifying nucleic acid fragments of bioparticles and connecting barcode molecular tags to amplified fragments for fragmentation of bioparticle DNA. Other enzymes may be co-separated, including but not limited to polymerases, transposases, ligases, proteinase K, DNase, and the like. Additional reagents may also include reverse transcriptases, enzymes with terminal transferase activity, primers, and oligonucleotides, and switching oligonucleotides (also referred to herein as "switching oligonucleotides" or "template switching oligonucleotides") that may be used for template switching. In some cases, template switching may be used to increase the length of cDNA. In some cases, template switching may be used to attach a predefined nucleic acid sequence to cDNA. In an example of template switching, cDNA may be generated from a template such as reverse transcription of a cell mRNA, wherein a reverse transcriptase with terminal transferase activity may add additional nucleotides, such as polyC, to the cDNA in a template-independent manner. The switching oligonucleotide may include a sequence complementary to an additional nucleotide, such as polyG. The other nucleotides (e.g., polyC) on the cDNA can be hybridized with the other nucleotides (e.g., polyG) on the switching oligonucleotide, so that the switching oligonucleotide can be used as a template by a reverse transcriptase to further extend the cDNA. The template switching oligonucleotide can include a hybridization region and a template region. The hybridization region can include any sequence that can hybridize with the target. In some cases, as previously described, the hybridization region includes a series of G bases to supplement the prominent C bases at the 3' end of the cDNA molecule. The series of G bases can include 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases or more than 5 G bases. The template sequence can include any sequence to be incorporated into the cDNA. In some cases, the template region includes at least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequence and / or functional sequence. The switching oligonucleotide can include deoxyribonucleic acid; ribonucleic acid; a modified nucleic acid containing 2-aminopurine, 2,6-diaminopurine (2-amino-dA), reverse dT, 5-methyl dC, 2'-deoxyinosine, super T (5-hydroxybutynyl-2'-deoxyuridine), super G (8-aza-7-deazaguanosine), locked nucleic acid (LNA), unlocked nucleic acid (UNA, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2' fluoro bases (e.g., fluoro-C, fluoro-U, fluoro-A, and fluoro-G), or any combination thereof.

[0153] In some cases, the toggle oligonucleotide can be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 2, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 , 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143 , 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168 , 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193 , 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or 250 nucleotides or longer.

[0154] In some cases, the toggle oligonucleotide can be at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 2, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 , 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143 , 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168 , 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193 , 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or 250 nucleotides.

[0155] Once the contents of the cell are released into its corresponding partition, the macromolecular components contained therein (e.g., macromolecular components of biological particles such as RNA, DNA or proteins) can be further processed in the partition. According to the method and system described herein, the macromolecular component content of a single biological particle can be provided with a unique identifier so that when characterizing those macromolecular components, they can be attributed to one or more biological particles derived from the same. The ability to attribute characteristics to a single biological particle or a biological particle group is provided by specifically assigning a unique identifier to a single biological particle or a biological particle group. A unique identifier, for example, in the form of a nucleic acid barcode, can be assigned or associated with a single biological particle or a biological particle population so as to mark or annotate the macromolecular components (and their characteristics) of a biological particle with a unique identifier. These unique identifiers can then be used to attribute the components and characteristics of the biological particle to a single biological particle or a biological particle group.

[0156] In some aspects, this is performed by isolating a single bioparticle or group of bioparticles together with a unique identifier, as described above (see Figure 2 ). In some aspects, a unique identifier is provided in the form of a nucleic acid molecule (e.g., an oligonucleotide) that includes a nucleic acid barcode sequence that can be linked to or otherwise associated with the nucleic acid content of a single biological particle or other components of a biological particle, particularly associated with fragments of those nucleic acids. The nucleic acid molecules are separated so that between nucleic acid molecules in a given partition, the nucleic acid barcode sequences contained therein are identical, but between different partitions, the nucleic acid molecules can and do have different barcode sequences, or at least a large number of different barcode sequences representing all partitions in a given analysis. In some aspects, only one nucleic acid barcode sequence can be associated with a given partition, but in some cases, there may be two or more different barcode sequences.

[0157] A nucleic acid barcode sequence can comprise about 6 to about 20 or more nucleotides within a nucleic acid molecule (e.g., oligonucleotide) sequence. In some cases, the length of the barcode sequence can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of the barcode sequence can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of the barcode sequence can be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. In some cases, the length of the barcode subsequence separated can be about 4 to about 16 nucleotides. In some cases, the barcode subsequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0158] The nucleic acid molecules separated together can also include other functional sequences for processing nucleic acids from the biological particles separated together. These sequences include, for example, targeted or random / universal amplification primer sequences for amplifying genomic DNA from a single biological particle in a partition, while connecting associated barcode sequences, sequencing primers or primer recognition sites, hybridization or detection sequences, such as for identifying the presence of a sequence or for pulling down barcoded nucleic acids or any of a number of other potential functional sequences. Other mechanisms for separating oligonucleotides together can also be used, including, for example, coalescence of two or more droplets, wherein one droplet contains an oligonucleotide, or oligonucleotides are microdistributed into partitions, such as droplets in a microfluidic system.

[0159] In an example, microcapsules such as beads are provided, each of which contains a plurality of barcoded nucleic acid molecules (e.g., barcoded oligonucleotides) described above releasably attached to the beads, wherein all nucleic acid molecules attached to a particular bead will contain the same nucleic acid barcode sequence, but wherein a plurality of different barcode sequences are represented across the population of beads used. In some embodiments, hydrogel beads, such as those comprising a polyacrylamide polymer matrix, are used as solid supports and vehicles for delivering nucleic acid molecules into partitions, as they are able to carry a large number of nucleic acid molecules and can be configured to release those nucleic acid molecules upon exposure to a particular stimulus, as described elsewhere herein. In some cases, a population of beads provides a diverse library of barcode sequences comprising at least about 1,000 distinct barcode sequences, at least about 5,000 distinct barcode sequences, at least about 10,000 distinct barcode sequences, at least about 50,000 distinct barcode sequences, at least about 100,000 distinct barcode sequences, at least about 1,000,000 distinct barcode sequences, at least about 5,000,000 distinct barcode sequences, or at least about 10,000,000 distinct barcode sequences or more. Additionally, each bead can be provided with a large number of attached nucleic acid (e.g., oligonucleotide) molecules. Specifically, the number of molecules of nucleic acid molecules comprising a barcode sequence on a single bead can be at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acid molecules, at least about 10,000,000 nucleic acid molecules, at least about 50,000,000 nucleic acid molecules, at least about 100,000,000 nucleic acid molecules, at least about 250,000,000 nucleic acid molecules, and in some cases at least about 1 billion nucleic acid molecules or more. The nucleic acid molecules of a given bead can comprise the same (or common) barcode sequence, different barcode sequences, or a combination of both. The nucleic acid molecules of a given bead can comprise multiple groups of nucleic acid molecules. Nucleic acid molecules of a given group can contain the same barcode sequence. The same barcode sequence can be different from the barcode sequence of another group of nucleic acid molecules.

[0160] In addition, when the bead population is separated, the resulting partitioned population can also comprise a different barcode library comprising at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences. In addition, each partition of the population can contain at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acid molecules, at least about 10,000,000 nucleic acid molecules, at least about 50,000,000 nucleic acid molecules, at least about 100,000,000 nucleic acid molecules, at least about 250,000,000 nucleic acid molecules, and in some cases at least about 1 billion nucleic acid molecules.

[0161] In some cases, it may be desirable to incorporate multiple different barcodes within a given partition, attached to a single or multiple beads within the partition. For example, in some cases, a mixed but known set of barcode sequences can provide greater assurance of identity in subsequent processing, for example, by providing a stronger barcode address or attribution to a given partition, as a duplicate or independent confirmation of a given partition output.

[0162] When a specific stimulus is applied to the beads, nucleic acid molecules (e.g., oligonucleotides) can be released from the beads. In some cases, the stimulus can be a light stimulus, such as by cutting a light labile bond that releases the nucleic acid molecule. In other cases, a thermal stimulus can be used, in which the increase in the ambient temperature of the beads will result in the cutting of a bond or other release of the nucleic acid molecule to form the beads. In still other cases, a chemical stimulus that cuts the bond between the nucleic acid molecule and the beads or otherwise causes the nucleic acid molecule to be released from the beads can be used. In one case, such compositions include the polyacrylamide matrix described above for encapsulating bioparticles, and can be degraded to release the connected nucleic acid molecules by exposure to reducing agents such as DTT.

[0163] In some aspects, systems and methods for controlled separation are provided. Droplet size can be controlled by adjusting certain geometric features in a channel architecture (e.g., a microfluidic channel architecture). For example, the expansion angle, width, and / or length of the channel can be adjusted to control droplet size.

[0164] Figure 4An example of a microfluidic channel structure for controlled separation of beads into discrete droplets is shown. The channel structure 400 may include a channel segment 402 that is connected to a reservoir 404 at a channel junction 406 (or intersection). The reservoir 404 may be a chamber. As used herein, any reference to a "reservoir" may also refer to a "chamber." In operation, an aqueous fluid 408 containing suspended beads 412 may be transported along the channel segment 402 to the junction 406 to meet with a second fluid 410 that is immiscible with the aqueous fluid 408 in the reservoir 404 to produce droplets 416, 418 of the aqueous fluid 408 flowing into the reservoir 404. At the junction 406 where the aqueous fluid 408 and the second fluid 410 meet, the droplets may be separated based on, for example, the hydrodynamic force at the junction 406, the flow rates of the two fluids 408, 410, the fluid properties, and certain geometric parameters of the channel structure 400 (e.g., w, h 0 , α, etc. A plurality of droplets can be collected in the reservoir 404 by continuously injecting an aqueous fluid 408 from the channel segment 402 through the junction 406.

[0165] The generated discrete droplets may contain beads (e.g., as in occupied droplet 416). Alternatively, the generated discrete droplets may contain more than one bead. Alternatively, the generated discrete droplets may not contain any beads (e.g., as in unoccupied droplet 418). In some cases, the generated discrete droplets may contain one or more biological particles, as described elsewhere herein. In some cases, the generated discrete droplets may include one or more reagents, as described elsewhere herein.

[0166] In some cases, aqueous fluid 408 can have a substantially uniform concentration or frequency of beads 412. Beads 412 can be delivered from separate channels ( Figure 4 The beads 412 are introduced into the channel segment 402 (not shown). The frequency of the beads 412 in the channel segment 402 can be controlled by controlling the frequency at which the beads 412 are introduced into the channel segment 402 and / or the relative flow rates of the fluids in the channel segment 402 and the individual channels. In some cases, the beads can be introduced into the channel segment 402 from multiple different channels and the frequency controlled accordingly.

[0167] In some cases, the aqueous fluid 408 in the channel segment 402 may include biological particles (e.g., Figure 1 and 2402). In some cases, aqueous fluid 408 can have a substantially consistent concentration or frequency of biological particles. Like beads, biological particles can be introduced into channel segment 402 from a separate channel. The frequency or concentration of biological particles in aqueous fluid 408 in channel segment 402 can be controlled by controlling the frequency at which biological particles are introduced into channel segment 402 and / or the relative flow rates of the fluids in channel segment 402 and the separate channels. In some cases, biological particles can be introduced into channel segment 402 from a plurality of different channels, and the frequency can be controlled accordingly. In some cases, a first separate channel can introduce beads and a second separate channel can introduce biological particles into channel segment 402. The first separate channel into which beads are introduced can be upstream or downstream of the second separate channel into which biological particles are introduced.

[0168] The second fluid 410 may include an oil, such as a fluorinated oil, that contains a fluorinated surfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets.

[0169] In some cases, the second fluid 410 may not be subjected to and / or directed to any flow into or out of the reservoir 404. For example, the second fluid 410 may be substantially stationary in the reservoir 404. In some cases, the second fluid 410 may be subjected to flow within the reservoir 404, but not into or out of the reservoir 404, such as by applying pressure to the reservoir 404 and / or being affected by the incoming flow of the aqueous fluid 408 at the junction 406. Alternatively, the second fluid 410 may be subjected to and / or directed to flow into or out of the reservoir 404. For example, the reservoir 404 may be a channel that directs the second fluid 410 from upstream to downstream, transporting the generated droplets.

[0170] The channel structure 400 at or near the junction 406 may have certain geometric features that at least partially determine the size of a droplet formed by the channel structure 400. The channel segment 402 may have a height h at or near the junction 406. 0 and width w. For example, channel segment 402 may include a rectangular cross-section that leads to a reservoir 404 having a wider cross-section (e.g., width or diameter). Alternatively, the cross-section of channel segment 402 may be other shapes, such as circular, trapezoidal, polygonal, or any other shape. The top and bottom walls of reservoir 404 at or near junction 406 may be inclined at an expansion angle α. The expansion angle α allows the tongue (a portion of the aqueous fluid 408 that leaves channel segment 402 at junction 406 and enters reservoir 404 before droplet formation) to increase depth and promote a reduction in the curvature of the droplet formed in the middle. The droplet size may decrease as the expansion angle increases. For the above-mentioned geometric parameter h 0 , w and α, the resulting droplet radius R can be predicted by the following equation d :

[0171]

[0172] For example, for a channel structure of w = 21 μm, h = 21 μm, and α = 3°, the predicted droplet size is 121 μm. In another example, for a channel structure of w = 25 μm, h = 25 μm, and α = 5°, the predicted droplet size is 123 μm. In another example, for a channel structure of w = 28 μm, h = 28 μm, and α = 7°, the predicted droplet size is 124 μm.

[0173] In some cases, the expansion angle α can range from about 0.5° to about 4°, about 0.1° to about 10°, or about 0° to about 90°. For example, the expansion angle can be at least about 0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 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°, or more. In some cases, the expansion angle can be at most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less. In some cases, the width w can be between about 100 micrometers (μm) and about 500 μm. In some cases, the width w can be between about 10 μm and about 200 μm. Alternatively, the width can be less than about 10 μm. Alternatively, the width can be greater than about 500 μm. In some cases, the flow rate of aqueous fluid 408 entering junction 406 can be between about 0.04 microliters (μL) / minute (min) and about 40 μL / min. In some cases, the flow rate of aqueous fluid 408 entering junction 406 can be between about 0.01 microliters (μL) / minute (min) and about 100 μL / min. Alternatively, the flow rate of aqueous fluid 408 entering junction 406 can be less than about 0.01 μL / min. Alternatively, the flow rate of aqueous fluid 408 entering junction 406 can be greater than about 40 μL / min, such as 45 μL / min, 50 μL / min, 55 μL / min, 60 μL / min, 65 μL / min, 70 μL / min, 75 μL / min, 80 μL / min, 85 μL / min, 90 μL / min, 95 μL / min, 100 μL / min, 110 μL / min, 120 μL / min, 130 μL / min, 140 μL / min, 150 μL / min, or more. At lower flow rates, such as a flow rate of about less than or equal to 10 μL / min, the droplet radius may not depend on the flow rate of aqueous fluid 408 entering junction 406.

[0174] In some cases, at least about 50% of the droplets generated may have a consistent size. In some cases, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the droplets generated may have a consistent size. Alternatively, less than about 50% of the droplets generated may have a consistent size.

[0175] The flux of droplet generation can be increased by increasing the number of generation points, such as increasing the number of junctions (e.g., junctions 406) between an aqueous fluid 408 channel segment (e.g., channel segment 402) and reservoir 404. Alternatively or additionally, the flux of droplet generation can be increased by increasing the flow rate of aqueous fluid 408 in channel segment 402.

[0176] Figure 5 An example of a microfluidic channel structure for increasing droplet generation flux is shown. The microfluidic channel structure 500 may include a plurality of channel segments 502 and a reservoir 504. Each channel segment of the plurality of channel segments 502 may be in fluid communication with the reservoir 504. The channel structure 500 may include a plurality of channel junctions 506 between the plurality of channel segments 502 and the reservoir 504. Each channel junction may be a droplet generation point. Figure 4 Any description of the channel segment 402 and its components of the channel structure 400 in 500 may correspond to any description of a given channel segment and its corresponding components of the plurality of channel segments 502 in the channel structure 500. Any description of the reservoir 404 and its components from the channel structure 400 may correspond to any description of the reservoir 504 and its corresponding components from the channel structure 500.

[0177] Each channel segment of the plurality of channel segments 502 may include an aqueous fluid 508 containing suspended beads 512. The reservoir 504 may include a second fluid 510 that is immiscible with the aqueous fluid 508. In some cases, the second fluid 510 may not be subjected to and / or directed to any flow into or out of the reservoir 504. For example, the second fluid 510 may be substantially stationary in the reservoir 504. In some cases, the second fluid 510 may be subjected to flow within the reservoir 504, but not into or out of the reservoir 504, such as by applying pressure to the reservoir 504 and / or being affected by the incoming flow of the aqueous fluid 508 at the junction. Alternatively, the second fluid 510 may be subjected to and / or directed to flow into or out of the reservoir 504. For example, the reservoir 504 may be a channel that directs the second fluid 510 from upstream to downstream, transporting the generated droplets.

[0178] In operation, an aqueous fluid 508 containing suspended beads 512 can be transported along the plurality of channel segments 502 to a plurality of junctions 506 to meet with a second fluid 510 in a reservoir 504 to produce droplets 516, 518. Droplets can be formed from each channel segment at each corresponding junction with the reservoir 504. At the junction where the aqueous fluid 508 and the second fluid 510 meet, the droplets can be formed based on, for example, the hydrodynamic forces at the junction, the flow rates of the two fluids 508, 510, the fluid properties, and certain geometric parameters of the channel structure 500 (e.g., w, h 0 , α, etc.), as described elsewhere in this article. Multiple droplets can be collected in the reservoir 504 by continuously injecting aqueous fluid 508 from the multiple channel segments 502 through the multiple junctions 506. Flux can be significantly increased with the parallel channel configuration of the channel structure 500. For example, the frequency of droplet generation of a channel structure with five inlet channel segments including aqueous fluid 508 is five times that of a channel structure with one inlet channel segment, provided that the fluid flow rates in the channel segments are substantially the same. The fluid flow rates in different inlet channel segments may be substantially the same or may not be substantially the same. According to the actual situation and the size of the reservoir allowed, the channel structure may have as many parallel channel segments as possible. For example, the channel structure can have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 500, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 5000 or more parallel or substantially parallel channel segments.

[0179] Geometric parameters, w, h 0 And α may or may not be consistent for each of the channel segments in the plurality of channel segments 502. For example, each channel segment may have the same or different widths at or near its corresponding channel junction with the reservoir 504. For example, each channel segment may have the same or different heights at or near its corresponding channel junction with the reservoir 504. In another example, the reservoir 504 may have the same or different expansion angles at different channel junctions with the plurality of channel segments 502. When the geometric parameters are consistent, advantageously, the droplet size can be controlled to be consistent even with increased flux. In some cases, when it is desired to have different droplet size distributions, the geometric parameters of the plurality of channel segments 502 may vary accordingly.

[0180] In some cases, at least about 50% of the droplets generated may have a consistent size. In some cases, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the droplets generated may have a consistent size. Alternatively, less than about 50% of the droplets generated may have a consistent size.

[0181] Figure 6 Another example of a microfluidic channel structure for increasing droplet generation flux is shown. The microfluidic channel structure 600 may include a plurality of channel segments 602 that are generally arranged in a circle around the perimeter of a reservoir 604. Each of the plurality of channel segments 602 may be in fluid communication with the reservoir 604. The channel structure 600 may include a plurality of channel junctions 606 between the plurality of channel segments 602 and the reservoir 604. Each channel junction may be a droplet generation point. Figure 2 Any description of the channel segment 402 and its components of the channel structure 400 in 600 may correspond to any description of a given channel segment and its corresponding components of the plurality of channel segments 602 in the channel structure 600. Any description of the reservoir 404 and its components from the channel structure 400 may correspond to any description of the reservoir 604 and its corresponding components from the channel structure 600.

[0182] Each channel segment of the plurality of channel segments 602 may include an aqueous fluid 608 containing suspended beads 612. The reservoir 604 may include a second fluid 610 that is immiscible with the aqueous fluid 608. In some cases, the second fluid 610 may not be subjected to and / or directed to any flow into or out of the reservoir 604. For example, the second fluid 610 may be substantially stationary in the reservoir 604. In some cases, the second fluid 610 may be subjected to flow within the reservoir 604, but not into or out of the reservoir 604, such as by applying pressure to the reservoir 604 and / or being affected by the incoming flow of the aqueous fluid 608 at the junction. Alternatively, the second fluid 610 may be subjected to and / or directed to flow into or out of the reservoir 604. For example, the reservoir 604 may be a channel that directs the second fluid 610 from upstream to downstream, transporting the generated droplets.

[0183] In operation, an aqueous fluid 608 containing suspended beads 612 can be transported to a plurality of junctions 606 along the plurality of channel segments 602 to meet with a second fluid 610 in a reservoir 604 to produce a plurality of droplets 616. Droplets can be formed from each channel segment at each corresponding junction with the reservoir 604. At the junction where the aqueous fluid 608 and the second fluid 610 meet, droplets can be formed based on factors such as the hydrodynamic force at the junction, the flow rate of the two fluids 608, 610, the fluid properties, and certain geometric parameters of the channel structure 600 (e.g., the width and height of the channel segment 602, the expansion angle of the reservoir 604, etc.), as described elsewhere herein. Multiple droplets can be collected in the reservoir 604 by continuously injecting the aqueous fluid 608 from the plurality of channel segments 602 through the plurality of junctions 606. Flux can be significantly increased with the substantially parallel channel configuration of the channel structure 600. Depending on the actual situation and the size of the reservoir allowed, the channel structure can have as many substantially parallel channel segments as possible. For example, channel structure can have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 5000 or more parallel or substantially parallel channel segments.Described multiple channel segments can be substantially evenly spaced, for example, around the edge or the periphery of reservoir.Alternately, the spacing of described multiple channel segments can be uneven.

[0184] The reservoir 604 may have an expansion angle α ( Figure 6 Each channel segment of the plurality of channel segments 602 may have a width w and a height h at or near each channel junction. 0 . Geometric parameters, w, h 0 α and α may or may not be consistent for each of the channel segments in the plurality of channel segments 602. For example, each channel segment may have the same or different widths at or near its corresponding channel junction with the reservoir 604. For example, each channel segment may have the same or different heights at or near its corresponding channel junction with the reservoir 604.

[0185] The reservoir 604 may have the same or different expansion angles at different channel junctions with the plurality of channel segments 602. For example, a circular reservoir (such as Figure 6The plurality of channel segments 602 may have a conical, dome-shaped, or hemispherical top plate (e.g., top wall) to provide the same or substantially the same expansion angle for each channel segment 602 at or near the plurality of channel junctions 606. When the geometric parameters are consistent, advantageously, the resulting droplet size can be controlled to be consistent even with increased flux. In some cases, when different droplet size distributions are desired, the geometric parameters of the plurality of channel segments 602 may be varied accordingly.

[0186] In some cases, at least about 50% of the droplets generated may have a consistent size. In some cases, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the droplets generated may have a consistent size. Alternatively, less than about 50% of the droplets generated may have a consistent size. The beads and / or bioparticles injected into the droplets may or may not have a consistent size.

[0187] Channel network, for example, as described above or elsewhere herein, can be coupled to suitable fluid assembly with fluid.For example, inlet channel section fluid is coupled to them and will be delivered to the appropriate material source of channel junction.These sources can comprise any one of multiple different fluid assembly, from being limited in the main structure of microfluidic device or being connected to its simple reservoir to the fluid conduit of delivery fluid such as from device outer source, manifold, fluid flow unit (for example, actuator, pump, compressor).Equally, outlet channel section (for example, channel section 208, reservoir 604 etc.) can be coupled to receiving container or conduit for separating cell with fluid for subsequent treatment.Again, this can be the reservoir limited in the microfluidic device main body or it can be the fluid conduit for separating cell delivery to subsequent process operation, instrument or assembly.

[0188] The methods and systems described herein can be used to greatly improve the efficiency of single-cell applications and / or other applications that receive droplet-based inputs. For example, after sorting occupied cells and / or cells of appropriate size, subsequent operations that can be performed can include the generation of amplification products, purification (e.g., by solid phase reversible immobilization (SPRI)), further processing (e.g., shearing, connecting functional sequences, and subsequent amplification (e.g., by PCR)). These operations can occur in batches (e.g., outside the partition). In the case where the partition is a droplet in an emulsion, the emulsion can be destroyed and the contents of the droplet are pooled for additional operations. Additional reagents that can be co-isolated with barcoded beads can include oligonucleotides that block ribosomal RNA (rRNA) and nucleases that digest genomic DNA from cells. Alternatively, rRNA removers can be applied during additional processing operations. The configuration of the construct generated by such methods can help minimize (or avoid) the sequencing of poly-T sequences during sequencing and / or sequencing the 5' end of the polynucleotide sequence. The amplification products, such as the first amplification product and / or the second amplification product, can be subjected to sequencing for sequence analysis. In some cases, amplification can be performed using a partial hairpin amplification (PHASE) method for sequencing.

[0189] A variety of applications require the assessment of the presence and quantification of different bioparticle or organism types within a bioparticle population, including, for example, microbiome analysis and characterization, environmental testing, food safety testing, epidemiological analysis, such as in tracking contamination, etc.

[0190] Method for forming a gel matrix

[0191] The methods and systems described herein can be used to generate discrete droplets including single bioparticles and polymer molecules, which are configured to be cross-linked under controlled conditions (e.g., click chemistry). In some cases, the cross-linked polymer can be a degradable matrix. In some cases, the cross-linked polymer can be a gel. In some cases, the cross-linked polymer can be a hydrogel matrix. In some cases, the cross-linked polymer can encapsulate biological samples, such as cells or nucleic acids. In some cases, additional reagents can penetrate into the cross-linked matrix. In some cases, the cross-linking between the polymer molecules can be cuttable, and after the matrix is ​​cut or degraded, the contents encapsulated in the matrix can be released. In some cases, the cross-linked matrix and / or the contents encapsulated therein can be barcoded.

[0192] Figure 7An example of a microfluidic channel structure 700 for co-separating biological particles and reagents to generate a cross-linked hydrogel matrix is ​​shown. The channel structure 700 can include channel segments 701, 702, 704, 706, and 708. Channel segments 701 and 702 are connected at a first channel junction 709. Channel segments 701, 702, 704, 706, and 708 are connected at a second channel junction 710.

[0193] In an example operation, the channel segment 701 can transport an aqueous fluid 712 containing a plurality of biological particles 714 along the channel segment 701 into the second junction 710. Alternatively or additionally, the channel segment 701 can transport beads (eg, gel beads). The beads can include barcode molecules.

[0194] For example, channel segment 701 can be connected to a reservoir including an aqueous suspension of biological particles 714. Upstream of second junction 710 and immediately before reaching it, channel segment 701 can meet channel segment 702 at first junction 709. Channel segment 702 can transport multiple reagents 715A (e.g., polymer molecules A) and 715B (e.g., polymer molecules B) suspended in aqueous fluid 712 along channel segment 702 to first junction 709. For example, channel segment 702 can be connected to a reservoir including reagents 715A and 715B. After first junction 709, aqueous fluid 712 in channel segment 701 can carry both biological particles 714 and reagents 715A and 715B toward second junction 710. In some cases, aqueous fluid 712 in channel segment 701 can contain one or more reagents, which can be reagents that are the same or different from reagents 715A and 715B. A second fluid 716 (e.g., oil) that is immiscible with the aqueous fluid 712 can be delivered from each of the channel segments 704 and 706 to the second junction 710. When the aqueous fluid 712 from channel segment 701 and the second fluid 716 from each of the channel segments 704 and 706 meet at the second channel junction 710, the aqueous fluid 712 can be separated into discrete droplets 718 in the second fluid 716 and flow out of the second junction 710 along the channel segment 708. The channel segment 708 can deliver the discrete droplets 718 to an outlet reservoir fluidly coupled to the channel segment 708, where the discrete droplets can be harvested.

[0195] The second fluid 716 may include an oil, such as a fluorinated oil, that contains a fluorinated surfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets 718 .

[0196] The generated discrete droplets may contain a single bioparticle 714 and / or one or more reagents in reagents 715A and 715B. In some cases, the generated discrete droplets may contain beads (not shown) carrying a barcode, such as other microfluidic structures described elsewhere herein. In some cases, the discrete droplets may be unoccupied (e.g., no reagents, no bioparticles). As shown below, click chemistry can be used to cross-link polymer molecules captured in the same discrete droplet to generate a hydrogel (e.g., 715A and 715B). In some cases, click chemistry can be used to generate a degradable hydrogel (e.g., 715A and 715B).

[0197] gel

[0198] As used herein, the term "gel" generally refers to a three-dimensional polymeric matrix; a hydrogel is an example of a gel. A gel can have both liquid and solid properties and can exhibit an organized material structure. A hydrogel can be a three-dimensional, hydrophilic, polymeric matrix configured to absorb / accommodate water or a biological fluid. In some cases, when water is a dispersion medium, a hydrogel can become swollen due to water. See, for example, Eur. Polym. J., 2015, 65: 252-67 and J. Adv. Res, 2015, 6: 105-21, each of which is incorporated herein by reference as a whole for all purposes. A hydrogel can take many forms. In some cases, a hydrogel can be a water-swollen, cross-linked polymeric network produced by a cross-linking reaction between monomers. In some cases, a hydrogel can be a polymeric material that retains water in its matrix but may not dissolve in water.

[0199] Hydrogels can be synthesized in many ways. In some cases, hydrogels can be synthesized by a one-step procedure, such as polymerization of multifunctional monomers and simultaneous crosslinking reactions. In some cases, hydrogels can be synthesized by multiple step procedures, for example, first polymerizing monomers and then performing crosslinking reactions by using orthogonal reactive groups that can respond to different conditions to allow a stepwise approach.

[0200] Hydrogel products can be classified based on their polymer composition (homopolymer hydrogel, copolymer hydrogel or multipolymer hydrogel), crosslinking type (chemical crosslinking or physical crosslinking), physical appearance (matrix, film or microsphere), network charge (nonionic, ionic, amphoteric or zwitterionic) and source (natural (e.g., chitosan) or synthetic (e.g., polyacrylamide)).

[0201] Hydrogels can be synthesized by techniques that can produce cross-linked polymers. In some cases, copolymerization / cross-linking free radical polymerization can be used to produce hydrogels by reacting hydrophilic monomers with multifunctional cross-linking molecules. This can be achieved by, for example, connecting polymer chains by chemical reactions, using ionizing radiation to generate backbone free radicals that can be used as cross-linking junctions for reorganization, or physical interactions such as entanglement, static electricity, and crystallite formation. Polymerization types can include bulk polymerization, solution polymerization, and suspension polymerization.

[0202] Suspension polymerization or dispersion polymerization can be used for water-in-oil or emulsion processes, sometimes referred to as "inverse suspension". In some cases, the monomers and initiator can be dispersed in an oil phase or a hydrocarbon phase as a homogeneous mixture. In some cases, two types of polymer molecules can be first produced, each of which has a reactive cross-linking moiety for cross-linking purposes. The two types of polymer molecules can then be encapsulated in an emulsion so that the two reactive cross-linking moieties can react and form cross-links between the two types of polymers, thereby completing the synthesis of the hydrogel.

[0203] In some cases, a hydrogel can be synthesized from a monomer, a polymerization initiator, and a cross-linking agent. After the polymerization reaction is complete, the formed hydrogel can be separated from the remaining starting materials and unwanted byproducts, etc. The length of the formed polymer can be controlled according to the desired properties of the hydrogel.

[0204] The types of polymerization used to synthesize the hydrogel may include, but are not limited to, graft polymerization, cross-linking polymerization, network formation of water-soluble polymers, and radiation cross-linking polymerization.

[0205] Polymerization can be initiated by initiators or free radical generating compounds such as benzoyl peroxide, 2,2-azo-isobutyronitrile (AIBN) and ammonium peroxodisulfate or by using UV, gamma or electron beam radiation.

[0206] In some cases, the hydrogels disclosed herein include polymers such as poly(acrylic acid), poly(vinyl alcohol), poly(vinyl pyrrolidone), poly(ethylene glycol), polyacrylamide, some polysaccharides, or any derivatives thereof. These polymers may be non-toxic, and the polymers may be used in various pharmaceutical and biomedical applications. Therefore, in some cases, the polymer may not need to be removed from the reaction system, thereby eliminating the need for a purification step after the hydrogel is formed.

[0207] The polymer can include polymer molecules of a particular length or range of lengths. The polymer molecules can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400 400, 450, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 2,000,000, 5,000,000, 10,000,000, 20,000,000, 100,000,000, 200,000,000, 500,000,000 or 1,000,000,000 skeletal atoms or molecules (e.g., carbon). The length of the polymer molecules can be up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500 In some embodiments, the present invention can include 0, 350, 400, 450, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 2,000,000, 5,000,000, 10,000,000, 20,000,000, 100,000,000, 200,000,000, 500,000,000 or 1,000,000,000 skeletal atoms or molecules (e.g., carbon).The polymer molecules can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500 00, 100, 000, 200, 000, 50, 000, 100, 000, 200, 000, 500, 000, 1,000, 000, 2,000, 000, 5,000, 000, 10,000, 000, 20, 000, 000, 100, 000, 000, 200, 000, 000, 500, 000, 000 or 1,000, 000, 000 monomer units (e.g., vinyl molecules or acrylamide molecules). The length of the polymer molecules can be up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500 00, 100, 000, 200, 000, 50, 000, 100, 000, 200, 000, 500, 000, 1,000, 000, 2,000, 000, 5,000, 000, 10,000, 000, 20, 000, 000, 100, 000, 000, 200, 000, 000, 500, 000, 000 or 1,000, 000, 000 monomer units (e.g., vinyl molecules or acrylamide molecules).

[0208] Click Chemistry

[0209] As used herein, the term "click chemistry" generally refers to the following reactions: modular, wide range, high yield, only generate harmless by-products such as by-products that can be removed by non-chromatographic methods, and have stereospecificity (but not necessarily enantioselectivity). See, for example, Angew. Chem. Int. Ed., 2001, 40 (11): 2004-2021, which is incorporated herein by reference for all purposes. In some cases, click chemistry can describe paired functional groups that can selectively react with each other under mild aqueous conditions.

[0210] An example of a click chemistry reaction can be the Huisgen 1,3-dipolar cycloaddition of azides and alkynes, i.e., the copper-catalyzed reaction of azides and alkynes to form a 5-membered heteroatom ring called 1,2,3-triazole. The reaction may also be referred to as Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), Cu(I) click chemistry, or Cu + Click chemistry. The catalyst of click chemistry can be a Cu(I) salt or a Cu(I) salt prepared in situ by reducing a Cu(II) reagent to a Cu(I) reagent with a reducing agent (Pharm Res. 2008, 25(10): 2216–2230). Known Cu(II) reagents for click chemistry may include, but are not limited to, Cu(II)-(TBTA) complexes and Cu(II)(THPTA) complexes. TBTA, i.e., tris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, also known as tris-(benzyltriazolylmethyl)amine, may be a stabilizing ligand for a Cu(I) salt. THPTA, i.e., tris-(hydroxypropyltriazolylmethyl)amine may be another example of a stabilizer for Cu(I). Other conditions can also be accomplished to construct 1,2,3-triazole rings from azides and alkynes using copper-free click chemistry, such as via strain-promoted azide-alkyne click chemistry (SPAAC, see, e.g., Chem. Commun., 2011, 47:6257-6259 and Nature, 2015, 519(7544):486-90), each of which is incorporated herein by reference in its entirety for all purposes.

[0211] The present disclosure also contemplates the use of click chemistry to produce chemical bonds that are not 1,2,3-triazoles. A series of such click chemistry reactions that can be used to prepare biocompatible gels are well known in the art. See, for example, Madl and Heilshorn, "Bioorthogonal Strategies for Engineering Extracellular Matrices", Advanced Functional Materials (Adv.Funct.Mater.) 2018, 28: 1706046, which is incorporated herein by reference.

[0212] An example of a copper-free click chemistry reaction that does not produce a 1,2,3-triazole bond that can be used in the compositions and methods of the present disclosure is the Inverse-electron demand Diels-Alder (IED-DA) reaction. (See, e.g., Madl and Heilshorn 2018.) As described elsewhere herein, in the IED-DA click chemistry reaction, the click chemistry functional group pair includes a tetrazine group and a trans-cyclooctene (TCO) group or a tetrazine group and a norbornene group. This reaction does not contain copper and produces a bond that includes a dihydropyridazine group instead of a 1,2,3-triazole.

[0213] Other specific biorthogonal click chemistry reactions that can be used in the compositions and methods of the present disclosure but produce chemical bonds other than 1,2,3-triazoles include Diels-Alder reactions between a pair of furan and maleimide functional groups, Staudinger ligations, and nitrile oxide cycloaddition reactions. These click chemistry reactions and others are well known in the art and are described, for example, in Madl and Heilshorn 2018.

[0214] Thus, in some embodiments, the copper-free click chemistry that can be used to form the cross-linked polymers of the present disclosure can be selected from: (a) strain-promoted azide / dibenzocyclooctyne-amine (DBCO) click chemistry; (b) inverse electron demand Diels-Alder (IED-DA) tetrazine / trans-cyclooctene (TCO) click chemistry; (c) inverse electron demand Diels-Alder (IED-DA) tetrazine / norbornene click chemistry; (d) Diels-Alder maleimide / furan click chemistry; (e) Staudinger ligation; and (f) nitrile oxide / norbornene cycloaddition click chemistry.

[0215] For example, Figure 7 The discrete droplets 718 shown in FIG. 7 can be subjected to Figure 8 The copper-catalyzed click chemistry conditions are shown in . Figure 8An example process of forming a hydrogel by click chemistry in an emulsion system is shown. Figure 8 As shown, emulsion systems 800, 802, and 804 can represent different stages of crosslinking of polymer molecules to form a hydrogel. Emulsion system 800 can include discrete droplets 808 (including water) immersed in an oil phase 810. Within the discrete droplets 808, two polymer molecules 812 and 814 can be separated together. Polymer molecule 812 can include a first crosslinking precursor comprising an unstable bond 816 (e.g., a disulfide bond) and a first click chemistry portion 818. Polymer molecule 814 can include a second click chemistry portion 820. In addition, in the oil phase 810, there can be other reagents such as reagent 822 (shown as a copper (II) reagent), which are required to promote the click chemistry reaction between the first click chemistry portion 818 and the second click chemistry portion 820, whether by itself or by its derivatives. Because reagent 822 is retained outside the discrete droplets 808, a click chemistry reaction generally does not occur within the discrete droplets 808.

[0216] In the emulsion system 802, some of the reagents in the reagents 822 can be infiltrated into the discrete droplets 808 by physical or chemical processes. In some cases, the reagent 822 becomes or is otherwise processed to become the reagent 824 (shown as a copper (I) reagent) in the discrete droplets 808. The conversion to the reagent 824 may require additional reagents (not shown, for example, a reducing agent such as sodium ascorbate). In these embodiments, the reagent 824 can be a reagent required to initiate a click chemistry reaction between the first click chemistry portion 818 and the second click chemistry portion 820. Once in proximity to both the first click chemistry portion 818 and the second click chemistry portion 820, the reagent 824 can initiate a click chemistry reaction, such as a Cu (I) -catalyzed azide-alkyne cycloaddition (CuAAC), see emulsion system 804.

[0217] like Figure 8 As shown in the emulsion system 804, in the presence of reagent 824, as a result of the click chemistry reaction between the first click chemistry moiety 818 and the second click chemistry moiety 820, a crosslink 826 connecting two polymer molecules 812 and 814 is formed through the newly formed moiety 828. Thus, a hydrogel including crosslinked polymer molecules 812 and 814 can be formed. If desired, the reagent 822 and / or 824 can be removed from the newly formed hydrogel. In some cases, a stimulus (e.g., a chemical, thermal, or light stimulus) is applied to the labile bond 816 to release the crosslink 826 and / or degrade the hydrogel.

[0218] Copolymers with click chemistry moieties

[0219] Scheme 1 below depicts an example synthetic pathway leading to the generation of a pair of polymer molecules including click chemistry moieties for subsequent copolymerization reactions.

[0220] Solution 1

[0221]

[0222] Monomer A can react with monomer B to produce polymer C. Monomer B can include a linker L1 between the polymerizable portion and the click chemistry portion CL1. By changing the reaction conditions, polymer C can include a repeatable unit of the formula shown in Scheme 1, because n (n is an integer greater than 1) repeating regular polymer unit segments are sandwiched by at least one polymer unit, the polymer unit includes the click chemistry portion CL1, and there will be a total of m (m is a positive integer) units including the click chemistry portion CL1. The relative ratio between the repeating regular polymer unit and the unit with the click chemistry portion can be controlled in a variety of ways, including changing the relative amounts of the corresponding monomers A and B. The molecular weight and length of polymer C can be controlled by chain termination conditions. Similarly, monomer A can react with monomer D to produce polymer E. Monomer D can include a linker L2 between the polymerizable portion and the click chemistry portion CL2. By varying the reaction conditions, polymer E can include repeatable units of the formula shown in Scheme 1, wherein n (n is an integer greater than 1) repeating regular polymer unit segments are sandwiched by at least one polymer unit comprising a click chemistry moiety CL2, and there will be a total of m (m is a positive integer) units comprising the click chemistry moiety CL2. The relative ratio between repeating regular polymer units and polymer units with click chemistry moieties can be controlled in a variety of ways, including changing the relative amounts of the corresponding monomers A and D. The molecular weight and length of polymer E can be controlled by chain termination conditions. The integers n and m are independent in each case in Scheme 1, as polymers C and E can have the same or different integers n and m.

[0223] The length and / or chemical composition of the joint L1 and L2 can vary, depending on the size of the hydrogel pore, the rigidity of the joint, the hydrophilicity of the joint, etc. In general, the joint L1 and L2 can include any chemical group compatible with the desired click chemistry reaction conditions, the desired polymerization conditions, and / or the desired cell bead conditions. Therefore, in some embodiments, the composition of the joint L1 and L2 can include a chemical group selected from the following: amine, amide, aryl, imide, carbonate, carbamate, dihydropyridazine, ester, ether, heteroaryl, hydrazone, oxime, phosphodiester, polyethylene glycol (PEG), square acid, thiazole, thiazolidine, thioether, triazole, or any combination thereof. In some embodiments, the composition of the joint L1 and L2 can each include alkyl, alkoxy, alkylamino, alkylaminoalkyl, alkoxyalkyl, arylalkyl, arylalkoxy, arylalkylamino, heteroarylalkyl, heteroarylalkoxy, heteroarylalkylamino, or any combination thereof.

[0224] In some embodiments, at least one of the linkers L1 and L2 includes a copper chelating chemical group. Since the chelating group effectively increases the copper ion concentration at the reaction site, the presence of a copper chelating group in the linker L1 or L2 adjacent to the click chemistry portion CL1 or CL2 can promote the acceleration of the click chemistry reaction. See, for example, Uttamapinant et al., "Fast, Cell-compatible Click Chemistry with Copper-chelating Azides for Biomolecular Labeling" Angew. Chem. Int. Ed. Eng., June 11, 2012; 51 (24): 5852-5856, which is incorporated herein by reference. In addition, the use of a copper chelating linker with a click chemistry portion such as an azide-pyridylmethyl group can allow the use of significantly reduced copper concentrations for polymer cross-linking reactions, as described in the methods of the present disclosure. In some embodiments, linkers L1 and L2 including copper chelating groups such as pyridylmethyl groups can allow the copper concentration in the click chemistry reaction to be reduced by at least 10%, at least 25%, at least 50%, or at least 75% or more. In turn, in cell beads and gel beads prepared using low copper reactions, the use of lower copper concentrations can greatly increase the yield of biomolecules, such as reducing RNA degradation. In some embodiments, the use of copper chelating groups in linkers can result in an increase in the amount of detected genes (e.g., in cell bead-based gene expression measurements) by at least 10%, at least 25%, at least 50%, or at least 75% or more. The incorporation of exemplary copper chelating groups azide-pyridylmethyl in the click chemistry methods of the present disclosure is further described in the examples.

[0225] In some cases, at least one of the linkers L1 and L2 further includes an unstable bond. In some embodiments, the linkers L1 and / or L2 may further include more than one unstable bond, including a bioorthogonal unstable bond. Exemplary types of unstable bonds that may be included in L1 and / or L2 may include chemically unstable bonds, thermally unstable bonds, photolabile bonds, enzymatically unstable bonds, or combinations thereof. More specific examples of unstable bonds that may be part of L1 and L2 may include disulfide bonds, ester bonds, carbamate bonds, vicinal diol bonds, Diels-Alder bonds, sulfone bonds, silyl ether bonds, glycosidic bonds, peptide bonds, or phosphodiester bonds. Unstable bonds may also be bonds that can be cleaved by nucleic acid targeting enzymes such as restriction enzymes.

[0226] In some embodiments, linkers L1 and L2 further include disulfide bonds. The ability to cleave disulfide bonds with the reducing agent dithiothreitol (DTT) is well known in the art. In the embodiments disclosed herein, incorporation of labile disulfide bonds in linkers L1 and L2 is further described in the examples.

[0227] However, in some cases, it is desirable that DTT be present in the reagent mixture of the cell bead-based reaction, but still be able to selectively cleave linkers L1 and L2. Therefore, in some embodiments, linkers L1 and L2 may further include an unstable bond that is a carbamate bond. The carbamate bond is unstable in the presence of DTT, but can be selectively cleaved by diethylenetriamine (DETA) and heat. In some embodiments, linkers L1 and L2 may further include an unstable bond that is a carbamate bond and do not include a disulfide bond. Exemplary unstable linkers including carbamate groups and methods for cleaving carbamates and uses in the cell bead-based methods of the present disclosure are further described in the examples.

[0228] In some cases, the linkers L1 and L2 may further include an unstable bond that is an enzymatically unstable bond. For example, the linkers L1 and L2 may include a polypeptide having a sequence that is specifically cleaved by a specific protease. A linker comprising a specific polypeptide sequence having a terminal propargyl moiety may be incorporated into the polymer, such as Fig.34A The propargyl groups can then undergo standard CuAAC click chemistry reactions with azide-modified linkers on the second polymer to produce a 1,2,3-triazole cross-linked gel matrix, as shown. Fig.34B This gel matrix cross-linked by a specific polypeptide sequence can then be selectively enzymatically degraded by exposure to a protease that is selective for the peptide bonds in the sequence. Fig.34B As shown, a protease, such as type II collagenase, can selectively cleave an 8-mer polypeptide sequence, such as GGRMSMPV, at the peptide bond between the M and S amino acid residues.

[0229] It is well known that specific proteases can selectively cleave specific peptide bonds in a polypeptide sequence. It is also contemplated that proteases used to degrade polymer crosslinks in gels do not cleave other peptides / proteins that may be present in a cell bead-based bioassay system, such as polymerases or reverse transcriptases. Exemplary proteases that are highly selective for specific polypeptide sequences and can be used in embodiments associated with selective degradation of hydrogel matrices in the presence of bioparticles and other macromolecular components are provided in Table 1 below.

[0230] Table 1

[0231]

[0232] For example, HRV 3C protease is a highly specific protease that cleaves between the Q and G residues of the "3C" polypeptide cleavage tag LEVLFQGP, and is commercially available as a "PreScission Protease" or "PSP". Another exemplary highly specific protease is enterokinase. Enterokinase is an intestinal enzyme that is normally involved in the protease cleavage of trypsin, which specifically cleaves the peptide bond after the K of the "EKT" recognition sequence DDDDK. Similarly, as described above, type II collagenase specifically cleaves the peptide bond between M and S in the sequence GGRMSMPV.

[0233] In some embodiments, the linkers L1 and L2 may include a peptide bond that can be selectively cleaved by a protease. In some embodiments, the linkers L1 and L2 may include a polypeptide that includes a peptide bond that can be selectively cleaved by a protease, optionally wherein the polypeptide has a sequence selected from GGRMSMPV, LEVLFQGP, and DDDDK. In some embodiments, the protease is selected from HRV3C protease, enterokinase, and type II collagenase.

[0234] As described elsewhere herein, a wide range of click chemistry reactions can be used to generate polymers for the compositions and methods of the present disclosure. Useful click chemistry reactions include well-known copper-catalyzed reactions such as CuAAC, and copper-free click chemistry reactions. Therefore, parts CL1 and CL2 can include any pair of chemical groups that undergo click chemistry reactions. In some cases, one click chemistry part in a click chemistry part (CL1 or CL2) includes an azide and another includes an alkyne. In some cases, one click chemistry part in a click chemistry part (CL1 or CL2) includes an azide and another includes a dibenzocyclooctyne (DBCO) group.

[0235] Both azide-alkyne CuAAC reaction and strain-promoted Cu-free azide-DBCO reaction produce click chemistry bonds including 1,2,3-triazole moieties. However, as described elsewhere herein, the present disclosure also contemplates gel compositions and methods for preparing the gel compositions, wherein linkers and associated click chemistry reactions are used to exclude bonds of 1,2,3-triazoles. Such alternative click chemistry reactions and resulting bonds are well known in the art. See, for example, Madl and Heilshorn, "Biological orthogonal strategies for engineering extracellular matrix", Advanced Functional Materials 2018, 28: 1706046, which are incorporated herein by reference. Therefore, in some cases, it is considered that the click chemistry reaction is a reverse electron demand Diels-Alder reaction, wherein one of the click chemistry moieties (CL1 or CL2) includes a tetrazine group, and another group includes a trans-cyclooctene (TCO) group or a norbornene group. In both cases, the use of tetrazines with TCOs or norbornenes, click chemistry reactions produce bonds involving dihydropyridazine groups rather than 1,2,3-triazoles.

[0236] Other click chemistry moieties and reactions that do not produce 1,2,3-triazole but can be used in the gel compositions and methods of making the gel compositions described herein include furan-maleimide Diels-Alder reactions. Thus, in some embodiments, one of the click chemistry moieties (CL1 or CL2) includes a furan moiety and the other includes a maleimide moiety.

[0237] In some cases, the integers n and m are selected based on the properties of the resulting polymers C and E. Such properties may include viscosity of the polymers before / after cross-linking, stability, pore size of the formed hydrogel, gelation rate, purity, purification procedures, compatibility of the click chemistry moiety with the polymerization conditions, procedures required to remove the initiator, etc.

[0238] Click chemistry conditions

[0239] like Figure 8 As shown, in some embodiments, the copper (II) species (reagent 822) is present in the oil phase outside the discrete droplet including the polymer to be cross-linked. However, in these embodiments, the copper (I) species (reagent 824) is the catalyst that enables the click chemistry reaction to occur inside the discrete droplet. In these cases, the copper (II) species initially present in the external oil phase can be exchanged into the aqueous phase inside the discrete droplet and then reduced by a reducing agent (e.g., sodium ascorbate) in the aqueous phase to produce the copper (I) species in the aqueous phase inside the discrete droplet.

[0240] In some cases, the exchange process can be shown in Scheme 2. To form copper (II) species in the oil phase, the first step can be an exchange reaction between a copper (II) acetate salt and a fluorinated carboxylic acid represented as Krytox-COOH, which includes a perfluorinated alkyl chain to prepare a compound / complex a stable suspension in the perfluorinated oil phase with an acidic carboxylic acid group to complex copper (II). Perfluorinated compounds or polymers, such as poly (perfluoro-propylene oxide) are known as A type of compound produced by DuPont.

[0241] Then Krytox-COO - The complex copper (II) salt can be further combined with the perfluorinated surfactant Krytox-PEG-Krytox to form an emulsion droplet of copper (II) inside the aqueous phase of the droplet. For the composition of the droplet so formed, the surfactant Krytox-PEG-Krytox is retained at the interface of the aqueous phase (inside) and the perfluorinated oil phase (outside), with the PEG component of the surfactant facing the inner aqueous phase and the perfluorinated arms pointing to the outer oil phase. In this way, an oil phase suspension of copper (II) species is formed, and the oil phase suspension copper (II) species is stable in the oil phase during the click chemistry reaction. In addition, when necessary, such as before or after the click chemistry reaction is completed, the bare copper (II) species and / or the oil phase suspension copper (II) species can be removed by filtration.

[0242] In some cases, the order in which the above reagents (copper (II) acetate, Krytox-COOH, and surfactant Krytox-PEG-Krytox) are mixed can be important. For example, direct mixing of all three components may not provide the desired suspension of copper (II) species in the oil phase. A stepwise procedure of mixing copper (II) acetate and Krytox-COOH first, then adding the surfactant, along with stirring / mixing, etc. can produce the desired suspension.

[0243] Solution 2.

[0244]

[0245] Fluorosurfactants having two fluorophilic tails and one hydrophilic head group (Formula I, hereinafter referred to as "triblock surfactants") can reduce coalescence of emulsion droplets and provide stability to emulsion systems including, for example, emulsion systems containing gel beads. In addition, fluorosurfactants having one fluorophilic tail and one hydrophilic head group (Formula II, hereinafter referred to as "diblock surfactants" or "diblock copolymers") can also be used to provide stability to emulsion systems. Both n and m are integers greater than 1. Krytox-PEG-Krytox is an example of a triblock surfactant.

[0246]

[0247] The second step can be to transport / phase transfer the dissolved copper (II) species into the droplets containing the polymer to be cross-linked. Various factors can affect the transport / phase transfer, including but not limited to the concentration of the corresponding ligand (e.g., THPTA or TBTA) in the oil phase and / or the water phase, other aqueous components in the water phase (e.g., surfactants, magnetic particles, solvents such as water, F-108), the type and amount of reducing agent used (disulfide, such as dithiothreitol (DTT) or sodium ascorbate, polymer, and the v / v ratio between oil phase and aqueous phase during preparation).

[0248] The third step can be to reduce the copper (II) species to copper (I) species inside the aqueous phase of the droplet. The reducing agent, such as sodium ascorbate, can be selected based on its chemical properties as a reducing agent and its compatibility with other parts of the polymer and / or linker groups and / or other reagents present inside the aqueous phase of the droplet. For example, when the linker group (e.g., click chemistry moiety CL1 or CL2) includes a disulfide bond, the use of DTT as a copper reducing agent may interfere with the integrity of the polymer or hydrogel because the disulfide bonds within the linker group can be cleaved during the reduction of the copper (II) species, thereby preventing the formation of the desired hydrogel.

[0249] The fourth step can be a click chemistry reaction catalyzed by the copper (I) species inside the droplet. In some cases, there can be at least one pair of polymers with two click chemistry moieties 818 and 820 inside the droplet for the click chemistry reaction to occur. In some cases, there can be multiple pairs of such polymers. Figure 7 During the depicted droplet formation process, the number of such polymer pairs inside one droplet can be controlled. As cross-linking is involved, there may be situations where one polymer cross-links with more than one other polymer to form a hydrogel.

[0250] Factors that may be considered during the click chemistry step may include, but are not limited to, the size of the droplets, the length / number / type / ratio of polymers encapsulated in each droplet, the ratio of reducing agent to copper(I) / copper(II) species inside the droplet aqueous phase, the ratio of copper(I) species to click chemistry moieties on the polymer, the role of the ligand (e.g., THPTA or TBTA), the time and temperature of the reaction, the presence or absence of external influences (e.g., shaking or vortexing, microwaves, etc.), dissolved oxygen, and how to separate unwanted reagents / byproducts after the click chemistry reaction is completed.

[0251] In some cases, click chemistry reactions can be run under inert gas conditions. For example, the reaction can be run under N2 In some cases, the addition of a reducing agent is increased to offset the side reaction of this oxygen or air oxidation of the copper (I) species. In some cases, instead of starting with a copper (II) species, a copper (I) species is added to an aqueous fluid as a catalyst for the azide-alkyne cycloaddition. In some cases, when the copper (I) species is initially added to an aqueous fluid as a catalyst for the azide-alkyne cycloaddition, an oxygen-free system is provided for the reaction.

[0252] In some cases, solvent exchange may be performed to remove unwanted reagents from the formed hydrogel.

[0253] In some cases, the hydrogel can be further transformed by adding additional reagents to the hydrogel or removing some reagents from the hydrogel.

[0254] In some cases, a biological sample (e.g., a cell or cell nucleus or nucleic acid) is encapsulated inside the pores of a hydrogel formed during a click chemistry reaction. In some cases, the biological sample can be modified or characterized inside the hydrogel by reacting the biological sample with a reagent delivered into the hydrogel (e.g., through the hydrogel pores).

[0255] In some cases, copper nanoparticles can be compounded with cells encapsulated inside the hydrogel. In these cases, the copper nanoparticles are used to catalyze click chemistry reactions to form hydrogels. Copper nanoparticles can be used as a substitute or supplement for copper (II) or copper (I) species. For example, cells can be compounded with copper nanoparticles (e.g., into droplets) before separation. The cells including copper nanoparticles are then separated (e.g., into droplets) using the click chemistry polymers described herein to generate cross-linked hydrogels. The use of cell-complexed copper nanoparticles can allow selective gelation of the hydrogel so that the click chemistry reaction is performed only in droplets including copper-complexed cells.

[0256] In some cases, during the cross-linking reaction between the cross-linking precursors, the unstable bonds (e.g., disulfide bonds) in the cross-linking precursors or the cross-links formed thereby remain intact (i.e., not broken). In some cases, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the unstable bonds can remain intact during the cross-linking reaction. In some cases, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the unstable bonds can remain intact during the cross-linking reaction.

[0257] In some cases, unstable bonds in the cross-links (e.g., disulfide bonds) can be broken by treatment with a reagent (e.g., a reducing agent such as DTT, TCEP, etc.), so that the biological sample encapsulated inside the hydrogel is released and / or the pores of the hydrogel expand, so that one or more reagents capable of reacting with the biological sample can now access the biological sample.

[0258] Fig. 9 A schematic diagram of an example hydrogel generated using click chemistry is shown. A first copolymer 901 includes an alkyne portion 902, and a second copolymer 904 includes an azide portion 905. The alkyne portion 902 may include a degradable linker (e.g., a disulfide bond 903). Alternatively or in addition, the azide portion 905 may include a degradable linker. The alkyne portion 902 may react with the azide portion 905 in the presence of Cu(I) to generate an interchain linker including a 1,2,3-triazole portion 906.

[0259] Methods for cell bead generation

[0260] The methods of the present disclosure may include generating one or more cell beads comprising one or more of the polymers disclosed herein. Fig.10 An example method for generating cell beads is shown. In this example, cells and polymers or gel precursors are mixed with immiscible fluids (e.g., oil) to generate multiple aqueous droplets, including droplets 1001 including cells 1002. As described herein, droplets 1001 may include charged species. Droplet 1001 is subjected to conditions sufficient to polymerize or gelate polymers or gel precursors to generate cell beads 1003 including cells 1002. Gelation may include any of the gelation mechanisms and polymers described herein, including those utilizing click chemistry reactions, as described elsewhere herein. In some cases, cell beads 1003 are subjected to treatment conditions sufficient to lyse cells 1002, releasing the components of the cells into the cell beads. In other embodiments, before polymers or gel precursors are polymerized or gelated to generate cell beads 1003, cells 1002 are lysed in droplets 901. In still other embodiments, cells 1002 are permeabilized before or after polymerization or gelation of polymers or gel precursors. The cell beads are collected in the aqueous phase to generate a plurality of cell beads 1004. The cell beads can be stored for further processing. In some cases, after the polymer or gel precursor is polymerized or gelled, the charged species can be attached to the cell beads. For example, the polymer or gel precursor can include one or more functional groups that promote the connection of the charged species after the polymerization or gelation of the polymer or gel precursor. In other embodiments, the polymer or gel precursor includes a functional group that includes a charged species that is incorporated into the cell beads during the polymerization or gelation of the polymer or gel precursor.

[0261] On the one hand, the present disclosure provides a method for generating cell beads including charged species. First, a partition including cells, polymerization or gel precursors and charged species from multiple cells can be generated. Next, the partition can be subjected to conditions sufficient to react the polymerization or gel precursor to generate a polymer or gel network, the polymer or gel network including the cell or its derivative, and the charged species, thereby generating a cell bead. The partition can be subjected to conditions sufficient to polymerize or gel the polymerization or gel of the polymerization or gel precursor. Conditions sufficient to polymerize or gel the polymerization or gel of the polymerization or gel precursor are described elsewhere herein. In certain embodiments, cells are lysed to release cell components into the cell bead. Cells can be lysed before polymerization or gel precursor polymerization or gelation, simultaneously with polymerization or gel precursor polymerization or gelation, or after polymerization or gel precursor polymerization or gelation. In other embodiments, the cells in the cell bead are not lysed, but permeabilized to allow access to the components in the nucleus.

[0262] On the other hand, the present disclosure provides a method for generating cell beads including charged species. First, a partition including a cell nucleus separated from a cell, a polymerization or gel precursor and a charged species can be generated. Next, the partition can be subjected to conditions sufficient to react the polymerization or gel precursor to generate a polymer or gel network, wherein the polymer or gel network includes the cell nucleus and the charged species, thereby generating a cell bead. The partition can be subjected to conditions sufficient to polymerize or gel the polymerization or gel of the polymerization or gel precursor. Conditions sufficient to polymerize or gel the polymerization or gel of the polymerization or gel precursor are described elsewhere herein. For example, a copper catalyst can be used to catalyze a click chemistry reaction, thereby generating a hydrogel. In certain embodiments, the cell nucleus is cracked to release the cell nucleus component into the cell bead. The cell nucleus can be cracked before polymerization or gel precursor polymerization or gelation, simultaneously with polymerization or gel precursor polymerization or gelation, or after polymerization or gel precursor polymerization or gelation. In other embodiments, the cell nucleus in the cell bead is not cracked, but is permeabilized to allow access to the cell nucleus component in the cell nucleus.

[0263] The charged species may be a positively charged species. The positively charged species may be a reagent comprising a positive charge. The positively charged species may include trimethylammonium. The positively charged species may be (3-acrylamidopropyl)-trimethylammonium. The charged species may be a negatively charged species. The negatively charged species may include phosphate. The charged species may be connected to a polymer or gel network. During polymerization, the charged species may be incorporated into a polymer or gel network. The cell beads may include one or more chemical crosslinkers. The chemical crosslinker may be a disulfide bond. The charged species may be connected to one or more chemical crosslinkers. The derivative of the cell may be a component (e.g., DNA, RNA, protein, etc.) from the cell. The method for generating the cell beads may include lysing the cells within the partition (e.g., droplet) to release the component from the cell. The component may be a nucleic acid. The nucleic acid may be DNA (e.g., genomic DNA) or RNA (e.g., mRNA, siRNA). The component may be a protein. The component may be a negatively charged component, such as DNA, RNA, or miRNA. The component may be a positively charged component, such as a protein. The component from the cell may interact with the charged species. Components from cells can be non-covalently linked to charged species.

[0264] In some embodiments, negatively charged components from or derived from cells (e.g., DNA) interact with positively charged species (e.g., ((3-acrylamidopropyl)-trimethylammonium) of cell beads (e.g., positively charged functional groups of cell bead polymers) through ionic interactions. In other embodiments, positively charged components from or derived from cells (e.g., proteins) interact with negatively charged species (e.g., phosphate) of cell beads (e.g., negatively charged functional groups of cell bead polymers) through ionic interactions. In still other embodiments, negatively charged components from or derived from cells (e.g., DNA) interact with positively charged species (e.g., ((3-acrylamidopropyl)-trimethylammonium) of cell beads (e.g., positively charged functional groups of cell bead polymers) and positively charged components from or derived from cells (e.g., proteins) interact with negatively charged species (e.g., phosphate) of cell beads (e.g., negatively charged functional groups of cell bead polymers) through ionic interactions. In some embodiments, the present invention relates to a cell bead. The cell bead is a cell bead having a negatively charged species (e.g., phosphate) of a functional group. Therefore, for example, due to the electrostatic interaction with the charged species of the cell bead, one or more components from the cell can be retained in the cell bead. The component from the cell can retain about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours or longer in the cell bead. The component from the cell can retain at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours or longer in the cell bead. The component from the cell can retain at most 1 hour, at most 2 hours, at most 3 hours, at most 4 hours, at most 5 hours, at most 6 hours, at most 12 hours, at most 24 hours, at most 48 hours or at most 72 hours in the cell bead.

[0265] On the one hand, the present disclosure provides a method for generating cell beads (e.g., cell beads including charged species) comprising a charged polymer or gel network. First, a partition comprising cells and polymer or gel precursors from a plurality of cells can be generated. Next, the partition can be subjected to conditions sufficient to react the charged polymer or gel precursor to generate a charged polymer or gel network, the charged polymer or gel network comprising the cell or its derivative, thereby providing a cell bead comprising a charged species. The reaction can change the net charge on the polymer or gel precursor, thereby generating a charged polymer or gel network. The reaction can change the net charge on the polymer or gel network, thereby generating a charged polymer or gel network.

[0266] The polymer or gel precursor can be positively charged. The polymer or gel precursor can include chitosan. The polymer or gel precursor can include polyethyleneimine (PEI). The polymer or gel precursor can be negatively charged. The polymer or gel precursor can include alginate. The derivative of the cell can be a component (e.g., DNA, RNA, protein, etc.) from the cell. The method for generating cell beads can include cracking cells in a partition (e.g., a droplet) to release components from the cell. The component can be a nucleic acid. The nucleic acid can be DNA (e.g., genomic DNA) or RNA (e.g., mRNA, siRNA). The component can be a protein. The component can be a negatively charged component, such as DNA, RNA or miRNA. The component can be a positively charged component, such as protein. The component from the cell can interact with a charged polymer or gel network. The component from the cell can be non-covalently attached to a polymer or gel network of a cell bead including a charged species. In some embodiments, a negatively charged component (e.g., DNA) from or derived from a cell interacts with a positively charged species of a cell bead (e.g., a positively charged polymer or gel network) through ionic interactions. In other embodiments, the positively charged components (e.g., proteins) from or derived from cells interact with the negatively charged species of cell beads (e.g., negatively charged polymers or gel networks) through ionic interactions. In still other embodiments, the negatively charged components (e.g., DNA) from or derived from cells interact with the positively charged species of cell beads (e.g., positively charged polymers or gel networks), and the positively charged components (e.g., proteins) from or derived from cells interact with the negatively charged species of cell beads (e.g., negatively charged polymers or gel networks). Therefore, for example, due to the electrostatic interaction with the charged species of cell beads, one or more components from cells may be able to be retained in the cell beads. For example, due to the interaction with the charged polymer or gel network, the components from cells may be able to be retained in the cell beads. The components from cells can be retained in the cell beads for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours or more. The components from the cells can be retained in the cell beads for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours or more. The components from the cells can be retained in the cell beads for up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 12 hours, up to 24 hours, up to 48 hours or up to 72 hours.

[0267] On the one hand, the present disclosure provides a method for generating cell beads including charged species. First, a partition including cells and polymerization or gel precursors from multiple cells can be generated. Next, the partition can be subjected to conditions sufficient to react the polymerization or gel precursor to generate a polymer or gel network, and the polymer or gel network includes the cells or their derivatives. Next, the charged species can be coupled to the polymer or gel network, thereby providing a cell bead including a charged species. The partition can be subjected to conditions sufficient to polymerize or gel precursors. Conditions sufficient to polymerize or gel precursors are described elsewhere herein. For example, a copper catalyst can be used to catalyze click chemistry reactions, thereby generating a hydrogel. In some cases, cells are cracked to release cell components. Cells can be cracked before polymerization or gel precursor polymerization or gelation, while polymerization or gel precursor polymerization or gelation, or after polymerization or gel precursor polymerization or gelation.

[0268] Polymer or gel network can be a degradable polymer or gel network, as described herein, so that the cell beads are degradable cell beads. Any number of cell beads can be generated by generating multiple partitions. In some cases, about 1, about 2, about 3, about 4, about 5, about 10, about 50, about 100, about 500, about 1000, about 5000, about 10000, about 20000, about 50000, about 100000 or more cell beads are generated to generate multiple cell beads. Cell beads can be separated together with barcode beads (e.g., gel beads) for analysis of cells or their components.

[0269] Compositions for cell analysis

[0270] Disclosed herein is a composition including a cell bead, the cell bead including a polymerization or cross-linked polymer network, the polymerization or cross-linked polymer network including cells or lysed cells generated by cells, wherein the polymerization or cross-linked polymer network is charged. The cell bead can include components from cells. The component can be a nucleic acid. The nucleic acid can be a DNA (e.g., genomic DNA) or an RNA (e.g., mRNA, miRNA). The component can be a protein. The polymer network can be positively charged. The polymer network can include chitosan. The polymer network can include PEI. The polymer network can be negatively charged. The polymer network can include alginate. The component from the cell can be retained in the cell bead for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours or longer. The component from the cell can be retained in the cell bead for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours or longer. Components from the cells can be retained within the cell beads for up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 12 hours, up to 24 hours, up to 48 hours, or up to 72 hours.

[0271] Also disclosed herein is a composition comprising a cell bead, the cell bead comprising a polymerized or cross-linked polymer network, the polymerized or cross-linked polymer network comprising a cell or a lysed cell generated by a cell and a charged species. The cell bead may include a component from the cell. The component may be a nucleic acid. The nucleic acid may be a DNA (e.g., genomic DNA) or an RNA (e.g., mRNA, miRNA). The component may be a protein. The charged species may be positively charged. The charged species may include trimethylammonium. The charged species may be (2-aminoethyl) trimethylammonium. The charged species may be (3-acrylamidopropyl) trimethylammonium. The charged species may be negatively charged. The charged species may include phosphate. The component from the cell may be retained in the cell bead for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours or more. The components from the cells can be retained in the cell beads for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours or more. The components from the cells can be retained in the cell beads for up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 12 hours, up to 24 hours, up to 48 hours or up to 72 hours.

[0272] Fig.11AAn example cell bead of the present disclosure is shown, which includes a positively charged species connected to a polymer or gel network. Cell bead 1101 includes a positively charged species 1103 connected to a polymer or gel network. Cell bead 1101 including positively charged species 1103 can be generated using any of the methods disclosed herein, the method comprising polymerization or gelation of a charged polymerization or gel precursor (e.g., a gel precursor including a charged functional group). Cell bead 1101 also includes negatively charged cell components 1102 (e.g., nucleic acids, such as DNA) from a single cell. Positively charged species 1103 interact with negatively charged cell components 1102, thereby retaining negatively charged cell components 1102 in cell bead 1101. Cell bead 1101 is stored, and over time, negatively charged cell components 1102 hardly diffuse outside the cell bead.

[0273] Fig. 11B An example cell bead of the present disclosure is shown, which includes a negatively charged species connected to a polymer or gel network. Cell bead 1111 includes a negatively charged species 1113 connected to a polymer or gel network. Cell beads 1111 including negatively charged species 1113 can be generated using any of the methods disclosed herein, the method comprising polymerization or gelation of a charged polymerization or gel precursor (e.g., a gel precursor including a charged functional group). Cell beads 1111 also include positively charged cell components 1112 (e.g., proteins or polypeptides including net positive charge regions) from a single cell. Negatively charged species 1113 interact with positively charged cell components 1112, thereby retaining positively charged cell components 1112 in cell beads 1111. Cell beads 1111 are stored, and positively charged cell components 1112 are hardly diffused outside the cell beads.

[0274] Fig. 12AAn example cell bead of the present disclosure is shown, which includes a positively charged species connected to a releasable chemical crosslinker. Cell bead 1201 includes a positively charged species 1203 connected to a releasable chemical crosslinker 1204 (e.g., a disulfide bond). Cell bead 1201 including a positively charged species 1203 connected to a releasable chemical crosslinker 1204 can be generated using any of the methods disclosed herein, the method comprising polymerization or gelation of a gel precursor including a charged functional group connected to a releasable chemical crosslinker. In other embodiments, cell bead 1201 including a positively charged species 1203 connected to a releasable chemical crosslinker 1204 is generated by polymerization or gelation of a gel precursor, and then the polymerized or gelled cell beads are functionalized with a charged functional group using a crosslinker. Cell bead 1201 also includes a negatively charged cell component 1202 (e.g., nucleic acid, such as DNA) from a single cell. The positively charged species 1203 interacts with the negatively charged cell components 1202, thereby retaining the negatively charged cell components 1202 in the cell beads 1201. The cell beads 1201 are stored, and over time, the negatively charged cell components 1202 hardly diffuse out of the cell beads.

[0275] Fig. 12B An example of a cell bead of the present disclosure is shown, the cell bead including a negatively charged species connected to a releasable chemical crosslinker. Cell bead 1211 includes a negatively charged species 1213 connected to a releasable chemical crosslinker 1204 (e.g., a disulfide bond). Cell bead 1211 including a negatively charged species 1213 connected to a releasable chemical crosslinker 1214 can be generated using any of the methods disclosed herein, the method comprising polymerization or gelation of a gel precursor including a charged functional group connected to a releasable chemical crosslinker. In other embodiments, cell bead 1211 including a negatively charged species 1213 connected to a releasable chemical crosslinker 1214 is generated by polymerization or gelation of a gel precursor, and then the polymerized or gelled cell bead is functionalized with a charged functional group using a crosslinker. Cell bead 1211 also includes a positively charged cell component 1212 from a single cell (e.g., a protein or polypeptide including a net positive charge region). The negatively charged species 1213 interacts with the positively charged cell components 1212, thereby retaining the positively charged cell components 1212 in the cell beads 1211. The cell beads 1211 are stored, and over time, the positively charged cell components 1212 hardly diffuse out of the cell beads.

[0276] The charged cell beads and charged hydrogels of the present disclosure (e.g., Fig.11A -B. Fig. 12A-B) may also include any cross-linked polymer as disclosed elsewhere herein (e.g., click chemistry polymers, such as Figure 8 middle).

[0277] Cell beads

[0278] On the one hand, the present disclosure provides methods and systems for generating cell beads, which can be used for processing different components from a single cell. Cell beads can be generated by methods as described herein, for example, by polymerizing molecular precursors (for example, polymers or gel precursors) in the partitions including cells or components from cells. Cell beads can include one or more different types of components from cells, including for example DNA (for example, gDNA, chromatin, etc.), RNA (for example, mRNA, miRNA), proteins and / or metabolites. Components can be included in cell beads and / or connected to cell beads. Cells can be encapsulated in polymers or gel matrixes and lysed cells in gels or polymer matrixes, and lysed cells or lysed cells make their components encapsulated in polymers or gel matrixes to generate cell beads when cells are encapsulated in polymers or gel matrixes. Polymers or gel matrixes can include one or more charged species configured to interact with components (for example, DNA, RNA, proteins, etc.) from cells.

[0279] Partitions for generating cell beads may include one or more reagents for performing one or more reactions. Species may include, for example, reagents for nucleic acid amplification or extension reactions (e.g., primers, polymerases, nucleotides, cofactors (e.g., ionic cofactors), buffers, etc., including those described herein), reagents for enzymatic reactions (e.g., enzymes, cofactors, substrates, buffers, etc.), reagents for nucleic acid modification reactions such as polymerization, ligation, or digestion, and / or reagents for template preparation.

[0280] Reagents can include reagents for minimizing nucleic acid damage produced by click chemistry reactions. For example, free radical scavengers can be added to partitions to reduce the risk of free radicals generated during click chemistry reactions causing damage to nucleic acids. In some cases, free radical scavengers include dimethyl sulfoxide (DMSO). DMSO can be added to partitions for generating cell beads at sufficient concentrations to prevent nucleic acid damage. In some embodiments, DMSO is added to partitions in an amount of at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10% or more.

[0281] One or more reagents within a partition can be attached to a precursor (e.g., a polymer or gel precursor). The reagent can be covalently attached to the precursor. The reagent can be reversibly or irreversibly attached to the precursor. The reagent can be attached to the precursor via an acrylamide moiety.

[0282] In some cases, the oligonucleotide can be connected to a precursor. The oligonucleotide connected to the precursor can be used for example to capture RNA and / or perform a reverse transcription reaction. The oligonucleotide can include a poly-T sequence or a poly-U sequence (e.g., can be a poly-T primer). In certain embodiments, the poly-T sequence is used for hybridizing with a poly-A sequence, such as from the mRNA of a cell. In certain embodiments, the poly-U sequence is used for hybridizing with a poly-A sequence, such as from the mRNA of a cell.

[0283] In some cases, oligonucleotides such as poly-T sequences can be linked to precursors (e.g., polymers) via an irreversible click chemistry reaction. In some embodiments, such click chemistry linking of oligonucleotides can be performed during cross-linking of the polymers that creates the gel matrix. For example, Fig.35 Depicted, propargylated poly-T oligonucleotides introduced into emulsion droplets along with polymers modified with azide and alkyne click chemistry groups are linked via CuAAC click chemistry, resulting in the formation of 1,2,3-triazole bonds with some of the azide-modified linker sites of the azide-modified polymer. Other sites form crosslinks with the alkyne-modified polymer, resulting in a gel matrix that includes covalently linked poly-T agents capable of capturing polyadenylated RNA transcripts.

[0284] Partitions for generating cell beads may include one or more particles (e.g., magnetic particles). One or more reagents in the partition may be connected to the particle. Reagents may be covalently connected to the particle. Reagents may be reversibly or irreversibly connected to the particle. Reagents may be connected to the particle by acrylamide moieties. In some cases, oligonucleotides may be connected to the particle. The oligonucleotides connected to the particle may be used, for example, to capture RNA and perform reverse transcription reactions. In certain embodiments, the particle (which is optionally a magnetic particle) includes an oligonucleotide connected thereto, and the oligonucleotide includes a poly-T sequence that can hybridize with, for example, a poly-A sequence from the mRNA of the cell.

[0285] The cells in the partition can be lysed as described herein, thereby releasing the components from the cells into the partition. The components can include various types of cellular components, including proteins, metabolites and / or nucleic acid molecules (e.g., DNA, RNA (e.g., messenger RNA) etc.). Alternatively or in addition, the cells in the partition can be permeabilized. Permeabilization can allow certain reagents, species, components, etc. to be transferred in and / or out of the cells with or without complete cell lysis. In certain embodiments, the cells are lysed or permeabilized before the cell beads are polymerized or gelled. In other embodiments, the cells are lysed or permeabilized simultaneously with the cell beads being polymerized or gelled. In certain embodiments, the cells are lysed or permeabilized after the cell beads are polymerized or gelled. In still other embodiments, the cells are not lysed or permeabilized when they are in the cell beads.

[0286] Reagents can be contained in partitions, including reagents connected to precursors, particles, etc., and can be used to perform one or more reactions on cells or components from or derived from cells. The reaction can be, for example, amplification, reverse transcription or deamination reactions. In some embodiments, the one or more reactions are performed before cell bead polymerization or gelation. In some embodiments, the one or more reactions are performed simultaneously with cell bead polymerization or gelation. In some embodiments, the one or more reactions are performed after cell bead polymerization or gelation. In some cases, oligonucleotides (e.g., primers) are used to perform reverse transcription reactions on messenger RNA from cells, thereby generating complementary DNA (cDNA). Reverse transcription can include adding additional nucleotides to cDNA, for example, polynucleotides such as polyC. In some cases, template switching can be performed to further extend cDNA. Template switching can attach one or more additional sequences to cDNA. In some cases, additional sequences can be used to promote nucleic acid extension / amplification and / or barcoding, as described herein. cDNA can be connected to precursors and / or particles. In some cases, oligonucleotides are used to capture messenger RNA (e.g., by hybridization) from cells before generating cell beads.

[0287] Fig.13An exemplary method for generating cDNA from cell mRNA and connecting the cDNA to a polymeric precursor is shown. Partition 1300 (e.g., an aqueous droplet in an emulsion) may include cells 1301 and oligonucleotides 1302 connected to a polymeric precursor 1310. In some embodiments, oligonucleotide 1302 includes any other additional sequence such as a poly-T sequence, a random N polymer, a targeted capture / primer sequence, and / or a functional sequence as described elsewhere herein. In some cases, partition 1300 further includes one or more reagents, such as reagents for one or more reactions to one or more components of a cell (e.g., reverse transcriptase, buffer, cofactor, etc.) or reagents 1310 for polymerizing or gelling a polymeric precursor. Partition 1300 may also optionally include a template switching oligonucleotide (not shown). Cell 1301 is lysed or permeabilized, thereby releasing or otherwise allowing access to multiple types of cell components, which include messenger RNA (mRNA) 1303 and genomic DNA (gDNA) 1305. Oligonucleotide 1302 can be used to perform reverse transcription (RT) of mRNA, thereby generating complementary DNA (cDNA) 1304 connected to polymeric precursor 1310. In some cases, a template switching reaction can be performed using a template switching oligonucleotide to, for example, attach additional sequences to the cDNA. The polymeric precursor 1310 including the cDNA can then be polymerized or gelled to produce a cell bead including cDNA 1304 and gDNA 1305. In some embodiments, the polymeric precursor 1310 is polymerized or gelled to form a cell bead including mRNA 1303 (which can be hybridized with the oligonucleotide 1302), and gDNA 1305 and cDNA 1304 are generated in the cell bead. In some embodiments, the oligonucleotide 1302 is releasably connected to the gel precursor 1310 by an unstable bond.

[0288] Fig.14An example method for capturing cell mRNA or generating cDNA using oligonucleotides attached to magnetic particles is shown. Partition 1400 (e.g., aqueous droplets in an emulsion) can include cells 1401, oligonucleotides 1402 attached to particles 1403 (e.g., beads or magnetic particles), and polymeric precursors 1410. In some embodiments, oligonucleotides 1402 include poly-T sequences, random N polymers, targeted capture / primer sequences, and / or any other additional sequences such as functional sequences described elsewhere herein. In some cases, partition 1400 further includes one or more reagents, such as reagents for one or more reactions to one or more components of the cell (e.g., reverse transcriptase, buffer, cofactor, etc.) or reagents 1410 for polymerizing or gelling polymeric precursors. Cells 1401 can be lysed or permeabilized, thereby releasing or otherwise allowing access to multiple types of cell components, which include mRNA 1404 and genomic DNA (gDNA) 1405. The mRNA 1404 is then subjected to conditions that allow it to hybridize with the oligonucleotide 1402 (e.g., through a poly-T sequence), thereby capturing the mRNA. In some embodiments, the hybridized mRNA 1404 is converted into cDNA. The polymer precursor 1410 can then be polymerized or gelled to generate a cell bead, which includes the mRNA 1404 (or cDNA) and gDNA 1405 connected to the particle. Thus, the captured mRNA 1404 (e.g., hybridized with the oligonucleotide 1402 coupled to the particle 1403) is incorporated into the cell bead. In some cases, the captured mRNA 1404 or cDNA can be purified from the partition 1400 and processed separately.

[0289] In certain embodiments, the partition is subjected to conditions sufficient to generate cell beads including one or more reagents. For example, the partition droplet including the polymer precursor connected to the reagent (for example, primer, nucleic acid molecule, etc.) can be polymerized or gelled so that the reagent is connected to a polymer or gel matrix (that is, connected to the cell beads). In some cases, the reagent is releasably connected to the gel precursor by an unstable bond (for example, a chemically unstable bond, a thermally unstable bond, or a photolabile bond). The reagent can be covalently connected to the cell beads. The reagent can be reversibly or irreversibly connected to the cell beads. The reagent can be connected to the surface of the gel beads. The reagent can be connected to the inside of the cell beads. In some cases, mRNA is connected to the cell beads. For example, the polymer precursor connected to the mRNA from the cell can be polymerized or gelled to generate the cell beads so that the mRNA is connected to the cell beads. In some cases, cDNA is connected to the cell beads. For example, the polymer precursor connected to the cDNA derived from the cell can be polymerized to generate the cell beads so that the cDNA is connected to the cell beads. In some cases, one or more oligonucleotides are connected to the cell beads. For example, a polymer precursor attached to an oligonucleotide can be polymerized or gelled to generate a cell bead such that the oligonucleotide is attached to the cell bead.

[0290] Fig.15 An example of generating a cell bead comprising an agent connected to a polymer matrix is ​​shown. A partition 1500 comprising a polymer precursor 1510 connected to nucleic acid molecules 1502 and 1503 (e.g., mRNA, cDNA, primer, etc.) can be subjected to conditions sufficient to polymerize the polymer precursor, thereby generating a cell bead 1511 comprising nucleic acid molecules 1502 and 1503 connected to a polymer matrix 1530. In some cases, the partition 1550 includes a first type of polymer precursor 1510 and a second type of polymer precursor 1520, and generates a cell bead 1511 comprising a copolymer 1530 of polymer precursors 1510 and 1520. In some cases, the nucleic acid molecule is connected to the first and / or second polymer precursor. For example, the nucleic acid molecule 1502 can be connected to the first type of polymer precursor 1510, and the nucleic acid molecule 1503 can be connected to the second type of polymer precursor 1520, and generates a cell bead 1511 comprising nucleic acid molecules 1502 and 1503 connected to a polymer matrix 1540. In some cases, nucleic acid molecule 1502 and nucleic acid molecule 1503 are identical. In some cases, nucleic acid molecule 1502 and nucleic acid molecule 1503 are the same type of molecule (e.g., mRNA or cDNA), but may contain different sequences. In other cases, nucleic acid molecule 1502 and nucleic acid molecule 1503 are different.

[0291] Macromolecular components (for example, nucleic acid molecules, proteins, etc.) are connected to the particles in cell beads or cell beads and can be used to prepare species for further processing.For example, nucleic acid molecules connected to cell beads or particles can be processed while remaining connected to cell beads or particles.After processing, nucleic acid can be released (for example, released into partitions) from cell beads and / or particles for analysis.In some cases, a type of cell component or its derivative (for example, mRNA, cDNA) is connected to the particles in cell beads or cell beads, while encapsulating but not connecting another type of cell component (for example, genomic DNA) may be useful.This may be useful in, for example, promoting the separate processing of various types of components.For example, after cell beads are formed, cell beads can be transferred to an aqueous solution and subjected to other processing as described herein.For example, cell beads can be subjected to reverse transcription in batches to generate cDNA (for example, with oligonucleotide hybridization connected to cell bead matrix or particles such as magnetic particles) from captured mRNA.

[0292] Isolate cell beads

[0293] Cell beads can be isolated together with nucleic acid barcode molecules and nucleic acid molecules (e.g., mRNA, cDNA, gDNA, etc.) of or derived from the cell beads can be barcoded as described elsewhere herein. In some embodiments, cell beads are co-isolated with beads carrying barcodes (e.g., gel beads) and nucleic acid molecules of or derived from the cell beads are barcoded as described elsewhere herein. An overview of an example method for generating droplets comprising cell beads and nucleic acid barcode molecules is schematically depicted in Fig.16A middle. Fig.16A The method described in includes three stages 1610, 1620 and 1630; each respective stage includes: (1) generating cell beads (1610); (2) solvent exchange and treatment of the cell beads (1620); and (3) co-isolation of the cell beads and barcodes for subsequent labeling (e.g., barcoding) of one or more components of the cell beads (or derived therefrom) (1630).

[0294] Continue to refer Fig.16A , stage 1610 includes providing oil 1601, polymer or gel precursor 1602, and cells or nuclei 1603 (e.g., cells, fixed cells, cross-linked cells, nuclei, permeabilized nuclei, etc.) to microfluidic chip 1604 for droplet generation. The polymer or gel precursor can be charged as described, for example, in FIGS. 11-12 . The microfluidic chip 1604 can further be provided with charged species such as those described elsewhere herein ( Fig.16A ) for co-separation. Fig. 16BAs described in detail in , the microfluidic chip 1604 includes a plurality of reservoirs including oil 1601, polymer or gel precursors 1602, and cells 1603. The microfluidic chip 1604 may also include one or more additional reservoirs (not shown) including one or more additional reagents. The polymer or gel precursors 1602 and cells 1603 flow from their reservoirs (e.g., by the action of an applied force, such as negative pressure through a vacuum or positive pressure through a pump) to a first channel junction and combine to form a water stream. This water stream then flows to a second channel junction, where oil 1601 is provided. The water stream provided from the first channel junction is immiscible with the oil 1601, resulting in a suspension of aqueous droplets in the oil 1605, which then flows to a reservoir for collection. The flow within the microfluidic chip 1604 may be controlled by any suitable method, including using one or more flow regulators in one or more channels, customizing the size of the microfluidic channels, etc., as described elsewhere herein. As Fig.16A and Fig. 16B As shown, the product includes droplets 1605 that include cells from cells 1603 and polymeric or gel precursor 1602. In some cases, at least some of the droplets in droplets 1625 include a single cell.

[0295] In some cases, droplet 1605 is subjected to conditions sufficient to lyse the cells or cell nuclei contained therein, thereby releasing the cell macromolecular components into droplet 1605. The macromolecular components (e.g., nucleic acids, proteins, etc.) may be subjected to one or more reactions for additional processing. The processing of macromolecular components is described in more detail elsewhere herein. In other embodiments, droplet 1605 is subjected to conditions sufficient to permeabilize the cell (or cell nucleus), thereby facilitating access to one or more macromolecular components of the cell (or cell nucleus) for further processing. Droplet 1605 is then subjected to conditions suitable for polymerizing or gelling the polymer or gel precursor 1602 in droplet 1605 to generate cell beads 1606.

[0296] continue Fig.16A , the droplet 1605 is then subjected to conditions suitable for polymerizing or gelling the polymeric or gel precursor 1602 in the droplet 1605, which generates a cell bead 1606 encapsulating the cell (or cell nucleus) 1603. When the resulting cell bead 1606 is suspended in the oil, stage 1620 is initiated, which includes a solvent exchange configured to resuspend the cell bead 1606 in the aqueous phase 1611. Additional details and examples regarding solvent exchange are provided elsewhere herein.

[0297] The resuspended aqueous cell beads 1611 can then be optionally processed 1612 in batches to prepare cell beads for analyzing one or more cell components. For example, in 1612, the cell beads 1611 can be subjected to conditions suitable for lysing or permeabilizing the cells (or nuclei) in the cell beads 1613, thereby releasing or otherwise allowing access to one or more cell components (e.g., nucleic acids, such as mRNA and gDNA, proteins, etc.). Separately or simultaneously with cell lysis, the cell beads (e.g., 1611 or 1613) are also subjected in batches to conditions that denature nucleic acids derived from cells (e.g., gDNA) associated with the cell beads 1611. The polymer matrix of the cell beads 1613 effectively hinders or prevents larger molecules such as nucleic acids and / or proteins from diffusing from the cell beads 1613. In addition, in the case where a charged species is introduced into the polymer matrix, the charge (e.g., positive charge) of the cell beads effectively prevents the diffusion of molecules (e.g., nucleic acids) with opposite charges. Cell beads 1613 are sufficiently porous to facilitate diffusion of denaturants into the cell bead matrix to contact nucleic acids within the cell beads 1613. In some cases, the cell beads (1611 or 1613) can then be subjected in batches to conditions suitable for one or more reactions of nucleic acids or other analytes derived from cells associated with the cell beads (1611 or 1613). For example, antibodies can be washed into and / or out of the resuspended cell beads (1611 or 1613). After processing 1612, the resulting cell beads 1613 are then collected 1614 and can be stored before stage 1630 is initiated.

[0298] In stage 1630, droplets are generated that include cell beads (1611 or 1613) and barcode beads 1622 (e.g., gel beads that include nucleic acid barcode molecules attached thereto). Fig.16A As shown, a microfluidic chip 1623 is provided with oil 1621, cell beads 1613, and barcode beads 1622, each barcode bead comprising a barcode sequence (eg, each bead comprising a unique barcode sequence). Fig. 16C An example microfluidic chip 1623 is shown. Fig. 16CAs shown, microfluidic chip 1623 includes a plurality of reservoirs including oil 1621, cell beads 1613 and barcode beads 1622 (e.g., gel beads). The chip also includes additional reservoirs 1627 and 1628, which can be used to provide additional reagents (e.g., reagents for nucleic acid amplification, reagents that can degrade or dissolve cell beads and / or gel beads, reagents that degrade the bonds between barcodes and gel beads, etc.). Cell beads 1613 and barcode beads 1622 flow from their reservoirs (e.g., by the action of applied force, such as by the negative pressure of a vacuum or by the positive pressure of a pump) to the first channel junction and form an aqueous mixture. The material from reservoirs 1627 and 1628 can also be provided to the aqueous mixture at the first channel junction.

[0299] Alternatively, cell beads and gel beads can be mixed before introducing microfluidic chip. In this case, the single reservoir of microfluidic chip 1623 includes a mixture of cell beads and gel beads. The ratio of cell beads to gel beads in the mixture can be changed to change the quantity of the droplets including single cell beads and single gel beads generated. The mixture of cell beads and gel beads can (for example, by the effect of the force applied, such as by the negative pressure of vacuum or by the positive pressure of pump) flow from the reservoir to the first channel junction, in some cases with the material from reservoir 1627 and / or 1628. As an alternative or in addition, cells can be mixed with gel beads. For example, a group of cells and cell beads can be mixed with gel beads, or a group of cells can be mixed with gel beads.

[0300] In some embodiments, the aqueous mixture including the cell beads 1613, the barcode beads 1621, and in some cases, additional reagents then flows to the second channel junction, to which the oil 1621 is provided. The aqueous mixture provided from the first channel junction is immiscible with the oil 1621, resulting in a suspension of aqueous droplets 1625 in the oil, which then flows to a reservoir for collection. The microfluidic chip may also include a reservoir 1629 that can receive excess oil from the flow emerging from the second channel. The flow within the microfluidic chip 1623 may be controlled by any suitable strategy, including the use of one or more flow regulators in the channels (see Fig. 16C and 16D ) or use one or more flow regulators connecting the channels, use various channels, customize the size of the channels, etc. Fig.16A and Fig. 16CAs shown, droplets 1625 include cell beads 1613 and barcode beads 1622 (e.g., gel beads), in addition to any other reagents provided by reservoirs 1627 and 1628. In some cases, at least some of droplets 1625 include a single cell bead and a single barcode bead (e.g., a single gel bead).

[0301] In the case where reagents that degrade or dissolve the cell beads 1613, barcode beads 1622 (e.g., gel beads), and / or the bond between the barcode and the barcode beads 1622 (e.g., gel beads) are present in the droplets, these reagents can release nucleic acids captured in the cell beads 1613 from the cell beads 1613 and / or release the barcode from the barcode beads 1622. The nucleic acid barcode molecules then interact with the released cell components (e.g., cell nucleic acids) to generate barcoded nucleic acid molecules for nucleic acid sequencing, as described elsewhere herein. In embodiments where the barcode beads (e.g., gel beads) are degraded or the nucleic acid barcode molecules are releasably attached to the barcode beads (e.g., gel beads), the barcoded cell components (e.g., barcoded cDNA or gDNA fragments) are not attached to the beads. In the case where a given droplet includes a single cell bead and a single barcoded bead including a nucleic acid barcode molecule (including a common barcode sequence), the barcoded cell component (or its derivatives) can be associated with the cell (or other biological sample such as bacteria or virus) of the given cell bead via the common barcode sequence.

[0302] Fig.16D Depicted are two example microfluidic reactions demonstrating the use of Fig.16A Methods and Fig. 16B and 16C The microfluidic device depicted in generates droplets 1625 that include cell beads and gel beads. Fig.16D (Panel A) shows a droplet containing cell beads and gel beads, while Fig.16D (Panel B) shows a droplet including cell beads comprising a magnetic material (eg, magnetic particles) and a gel bead.

[0303] Partitions comprising barcode beads (e.g., gel beads) associated with barcode molecules and beads (e.g., cell beads) encapsulating cellular components such as cellular nucleic acids can be used for component analysis as described in U.S. Patent Publication No. 2018 / 0216162, which is incorporated herein by reference in its entirety for all purposes. Fig.17The generation of partitions including barcode beads and cell beads is schematically depicted. Cell beads are generated by encapsulating cells in a polymer matrix to form cell beads in operation 1701. Cells are then lysed in operation 1702 so that nucleic acids and other components of the cells are released from the cells and trapped by the cell bead polymer matrix. Cell beads are then treated in operation 1703 under conditions suitable for, for example, digesting proteins and / or denaturing nucleic acids (e.g., by alkaline reagents). Cell beads can then be washed and separated for further processing.

[0304] Computer Systems

[0305] The present disclosure provides computer systems programmed to implement the methods of the present disclosure. Fig.18 A computer system 1801 is shown that is programmed or otherwise configured to (i) control a microfluidic system during droplet formation, such as the rate at which each component is added at different channels, (ii) control reaction conditions for click chemistry reactions inside droplets, and (iii) perform sequencing applications. Computer system 1801 can adjust various aspects of the present disclosure, such as adjusting the rate at which various reagents, such as reducing agents, are added, and adjusting the flow rate of fluid in one or more channels in a microfluidic structure when droplets are formed. Computer system 1801 can be an electronic device of a user or a computer system remotely located relative to the electronic device. The electronic device can be a mobile electronic device.

[0306] The computer system 1801 includes a central processing unit (CPU, also referred to herein as a "processor" and "computer processor") 1805, which may be a single-core or multi-core processor or multiple processors for parallel processing. The computer system 1801 also includes a memory or memory location 1810 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 1815 (e.g., a hard disk), a communication interface 1820 (e.g., a network adapter) for communicating with one or more other systems, and peripheral devices 1825, such as cache, other memory, data storage devices, and / or electronic display adapters. The memory 1810, the storage unit 1815, the interface 1820, and the peripheral devices 1825 communicate with the CPU 1805 via a communication bus (solid lines) such as a motherboard. The storage unit 1815 may be a data storage unit (or data repository) for storing data. The computer system 1801 may be operably coupled to a computer network ("network") 1830 by means of the communication interface 1820. The network 1830 may be the Internet, the internet and / or an extranet or an intranet and / or an extranet in communication with the Internet. The network 1830 is a telecommunications network and / or a data network in some cases. The network 1830 may include one or more computer servers that may implement distributed computing such as cloud computing. In some cases, the network 1830 may implement a peer-to-peer network with the aid of the computer system 1801, and the peer-to-peer network may implement coupling a device to the computer system 1801 to act as a client or server.

[0307] The CPU 1805 may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location such as the memory 1810. The instructions may be directed to the CPU 1805, which may then program or otherwise configure the CPU 1805 to implement the methods of the present disclosure. Examples of operations performed by the CPU 1805 may include fetching, decoding, executing, and writing back.

[0308] CPU 1805 may be part of a circuit such as an integrated circuit. One or more other components of system 1801 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0309] Storage unit 1815 can store files, such as drivers, libraries, and saved programs. Storage unit 1815 can store user data, such as user preferences and user programs. Computer system 1801 may include one or more additional data storage units in some cases, and the one or more additional data storage units are located outside computer system 1801, such as located on a remote server that communicates with computer system 1801 via an intranet or the Internet.

[0310] Computer system 1801 can communicate with one or more remote computer systems via network 1830. For example, computer system 1801 can communicate with a remote computer system of a user (e.g., an operator). Examples of remote computer systems include personal computers (e.g., portable PCs), tablets or tablet PCs (e.g., iPad, GalaxyTab), phones, smartphones (e.g. iPhone, Android enabled devices, ) or a personal digital assistant. A user can access the computer system 1801 via the network 1830.

[0311] The methods described herein may be implemented by machine (e.g., computer processor) executable code stored in an electronic storage location (e.g., memory 1810 or electronic storage unit 1815) of computer system 1801. Machine executable or machine readable code may be provided in the form of software. During use, the code may be executed by processor 1805. In some cases, the code may be retrieved from storage unit 1815 and stored on memory 1810 for access by processor 1805 at any time. In some cases, electronic storage unit 1815 may be excluded and machine executable instructions may be stored on memory 1810.

[0312] The code may be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or may be compiled during runtime. The code may be supplied in a programming language that may be selected to enable execution of the code in a pre-compiled or as-compiled manner.

[0313] Various aspects of systems and methods such as computer system 901 provided herein can be embodied in programming. Various aspects of technology can be regarded as "products" or "articles" generally in the form of machine (or processor) executable code and / or associated data, which are performed on a type of machine-readable medium or embodied in the type of machine-readable medium. Machine executable code can be stored on electronic storage units such as memory (e.g., read-only memory, random access memory, flash memory) or hard disks. "Storage" type media can include any or all tangible memories of computers, processors, etc. or their associated modules, such as various semiconductor memories, tape drives, hard disk drives, etc. that can provide non-temporary storage for software programming at any time. All or part of the software can sometimes communicate through the Internet or various other telecommunication networks. Such communication, for example, can realize loading software from one computer or processor into another computer or processor, such as loading from a management server or host computer into a computer platform of an application server. Thus, another type of medium that can carry software elements includes light waves, electric waves, and electromagnetic waves, such as those used across physical interfaces between local devices over wired and optical landline networks and over various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., can also be considered as mediums that carry software. As used herein, unless restricted to non-transitory, tangible "storage" media, terms such as computer or machine "readable media" refer to any medium that participates in providing instructions to a processor for execution.

[0314] Thus, machine-readable media such as computer executable code may take many forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, such as any storage device in any computer, such as any storage device that can be used to implement the databases shown in the accompanying drawings, etc. Volatile storage media include dynamic memory, such as the main memory of such computer platforms. Tangible transmission media include coaxial cables; copper wire and fiber optics, including wires that include buses within a computer system. Carrier transmission media may take the form of electrical or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer-readable media include, for example: a floppy disk, a floppy disk, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD or DVD-ROM, any other optical medium, a punch card, a paper tape, any other physical storage medium with a pattern of holes, a RAM, a ROM, a PROM and an EPROM, a flash-EPROM, any other memory chip or memory cartridge, a carrier wave carrying data or instructions, a cable or link carrying such a carrier wave, or any other medium from which a computer can read program code and / or data. Many of these forms of computer-readable media may involve carrying one or more sequences of one or more instructions to a processor for execution.

[0315] The computer system 1801 may include or communicate with an electronic display 1835 that includes a user interface (UI) 1840 for providing, for example, the extent of hydrogel formation and the swelling ratio of the hydrogel. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0316] The methods and systems of the present disclosure may be implemented by one or more algorithms. The algorithms may be implemented by software when executed by the central processing unit 1805. The algorithms may, for example, perform sequencing and adjust the addition of various reagents according to the extent of certain reactions.

[0317] The devices, systems, compositions and methods disclosed herein can be used for various applications, such as processing a single analyte (e.g., RNA, DNA or protein) or multiple analytes (e.g., DNA and RNA, DNA and protein, RNA and protein or RNA, DNA and protein) to form a single cell. For example, biological particles (e.g., cells or cell beads) are separated in partitions (e.g., droplets), and multiple analytes from the biological particles are processed for subsequent processing. The multiple analytes can be from a single cell. For example, this can enable simultaneous proteomic, transcriptomic and genomic analysis of cells.

[0318] Example 1: Synthesis of Monomers and Polymers with Click Chemistry Moieties

[0319] In some cases, carboxylic acid groups are introduced into the polymer as anchors to attach click chemistry moieties / precursors. As shown in Scheme 3, acid-containing polymer 1C can be prepared by reacting monomer 1A with monomer 1B in the presence of an initiator. The integers m and n are greater than 1.

[0320] Solution 3

[0321]

[0322] When about 1 wt % of monomer 1B relative to monomer 1A is used in the presence of about 1.6 M NaF (NaF:total monomer ratio is about 1:1) and a thermal initiator (e.g., 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, VA-044 from Wako, about 0.1 wt %) at a temperature of about 30° C. to about 50° C., the number average molar mass (M) can be obtained. n ) is about 156K, where the polydispersity index (MW / M n ) is about 1.786.

[0323] According to Scheme 4A, polymer 1C can be coupled with propargylamine 1D to obtain polymer 1E, which can carry multiple click chemistry moieties (alkynes). The coupling agent can be any coupling agent that forms an amide bond from an acid and an amine. For example, EDCI, HOBt or HATU. The reaction can be carried out at a controlled pH, such as about pH 5.0 to about pH 9.0, about pH 6.0 to about pH 8.0, about pH 6.5 to about pH 7.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0, about pH 8.5 or about pH 9.0.

[0324] Option 4A

[0325]

[0326] Alternatively, polymer 1E can be formed from monomer 1A and monomer 1F in the polymerization reaction shown in Scheme 5. Monomer 1F can be formed by a coupling reaction between monomer 1A and propargylamine 1D in the presence of a coupling agent similar to the coupling reagent used in Scheme 4. In some cases, polymer 1E can be formed at about 1.5 wt % of monomer 1F relative to monomer 1A at 30° C. in the presence of about 0.1 wt % AIBN and 1.2 M NaF.

[0327] Solution 5

[0328]

[0329] The synthesis of azide-containing polymers can also take at least two different routes. One route similar to Scheme 4A for propargyl-containing polymers is to couple the carboxylic acid of polymer 1C to azide-functional primary amine 1DD using an amide coupling agent to provide azide-containing polymer 1EE, as shown in Scheme 4B.

[0330] Option 4B

[0331]

[0332] Alternatively, the primary amine bearing the azide functionality can be a p-azidopicolyl compound 1DP, which can be coupled to polymer 1C using an amide coupling agent to provide p-azidopicolyl-containing polymer 1EF as shown in Scheme 4C.

[0333] Option 4C

[0334]

[0335] like Fig.29 As depicted and described elsewhere herein, the p-azidopicolyl functional group is capable of chelating Cu(I) / Cu(II), thereby facilitating copper-catalyzed click chemistry crosslinking between the azidopicolyl and corresponding alkyne linkers in the presence of significantly lower concentrations of copper ions. As demonstrated in Example 11, the use of lower copper concentrations results in improved biocompatibility (e.g., reduced RNA degradation), which greatly improves gene expression analysis and other bioassays performed in the presence of these crosslinked polymers.

[0336] In addition, as described elsewhere herein, in some embodiments, crosslinking of polymers can be performed using copper-free click chemistry. For example, copper-free strain-promoted azide-alkyne click chemistry (SPAAC) (see, e.g., Chemical Communications, 2011, 47: 6257-6259 and Nature, 2015, 519(7544): 486-90) can be used, wherein an azide-modified linker is reacted with a dibenzocyclooctyne-amine (DBCO)-modified linker to form a Fig.30 Click chemistry shown. The azide-modified linker 1EE attached to a polyacrylamide polymer can be used as described above in Scheme 4B.

[0337] DBCO-modified linkers attached to polyacrylamide 1EG can be prepared using the sulfonated DBCO-analog 1DG as shown in Scheme 4D.

[0338] Solution 4D

[0339]

[0340] Another approach is to polymerize monomer 1A with azide-containing acrylamide. The common point of both syntheses may be the amine reagent 1H, as shown in Scheme 6. The amine reagent 1H can be coupled with polymer 1C to give the azide-containing polymer 1J. Additionally, the amine reagent 1H can be coupled with acryloyl chloride to produce monomer 1I, which can be polymerized with monomer 1A to give the azide-containing polymer 1J.

[0341] Solution 6

[0342]

[0343] Propargyl-containing monomer 1M can be synthesized as shown in Scheme 7A. Propargyl alcohol can be reacted with carbonyl-diimidazole to give propargylating agent 1K. Monopropargylation of cystamine 1L with propargylating agent 1K can provide monopropargylated cystamine 1KL, which can be further acylated on the free amine to provide disulfide-linked propargylated monomer 1M. Monomer 1M is a degradable alkyne-containing monomer.

[0344] Scheme 7A

[0345]

[0346] Alternatively, a propargyl-containing monomer 1Q that does not contain a disulfide bond can be synthesized as shown in Scheme 7B. However, monomer 1Q contains an unstable carbamate bond. As described elsewhere herein, monomer 1Q can be used to form a cross-linked polymer that does not degrade in the presence of DTT but can selectively degrade in the presence of DETA and heat.

[0347] Option 7B

[0348]

[0349] The synthesis of Scheme 7B is essentially the same as Scheme 7A, however the reagent 1,6-dihexylamine 1R rather than cystamine 1L is reacted with the propargylating agent 1K to provide the monopropargylated hexylamine carbamate 1KR. The free amine of 1KR can then be further acylated to provide the carbamate-linked propargylated monomer 1Q.

[0350] Azide-containing monomers 1N can be prepared from p-azidoaniline by acylation as shown in Scheme 8.

[0351] Solution 8

[0352]

[0353] Each of monomers 1M, 1N can be polymerized with monomer 1A to produce a corresponding polymer with a click chemistry moiety.

[0354] Experiment 1: Preparation of copper (II) reagent in oil phase.

[0355] 1) Suspension of copper (II) salt. At room temperature, about 2.50 mM Krytox-COOH and about 1.25 mM Cu(OAc) 2 Stir in HFE-7500 for about 48 hours.

[0356] 2) Preparation of GB oil. GB oil can be prepared by mixing about 2.50 mM bis-Krytox-ethylene glycol-polymer (Krytox-PEG-Krytox or BKEP or Formula I) and about 0.25 mM Krytox in a solvent HFE-7500 engineered oil. GB oil can be pre-prepared in batches.

[0357] 3) Prepare the oil phase solution CB oil. The suspension of copper (II) salt and GB oil can be combined in a ratio of v / v=1:1, and the resulting mixture can be stirred at room temperature for about 1 hour and filtered through a 0.22 μm PES filter to remove insoluble copper (II) salt. The filtrate is a CB oil including HFE-7500 containing 1.25 mM BKEP, about 1.375 mM Krytox-COOH and about 0.625 mM copper (II). The concentration of copper (II) in the water layer can be analyzed using UV-vis absorption spectroscopy (maximum absorption wavelength at about 286 nm).

[0358] Experiment 2: Preparation of aqueous solution.

[0359] A stock solution 1 of an aqueous solution can be prepared by mixing a polymer pair of an azide-containing and an alkyne-containing polymer (3.5 w / v%), F-108 (0.5 w / v%), magnetic particles (0.12 w / v%), THPTA (0.25 mM) in water.

[0360] A stock solution 2 of the aqueous phase solution can be prepared by mixing a polymer pair of an azide-containing and an alkyne-containing polymer (3.5 w / v %), F-108 (0.5 w / v %), magnetic particles (0.12 w / v %), THPTA (0.25 mM) and sodium ascorbate (156 mM) in water.

[0361] Experiment 3: Click chemistry and gelation in the absence of cells.

[0362] To prepare an emulsion of discrete droplets, a method similar to Figure 7 . Specifically, stock solution 1 (30 μL, from Experiment 2) can be fed through channel 701; stock solution 2 (40 μL, from Experiment 2) can be fed through channel 702; and CB oil (200 μL, from Experiment 1) can be fed through channel 704. A collection of water-in-oil emulsions of droplets can be obtained in the collection well. The emulsion of droplets can be kept in the well (with lid) for about 60 minutes. Subsequent solvent exchange can convert the oil phase into the water phase. Gelation can be observed with the naked eye and under a microscope. The swelling ratio of the gel can be measured by comparing the size data between the monodispersity in the water phase (100 minutes) and the monodispersity in the NaOH phase (5 minutes) under a microscope.

[0363] Experiment 4: Click chemistry and gelation in the presence of cells.

[0364] To prepare an emulsion of discrete droplets, a method similar to Figure 7 . Specifically, stock solution 1 (30 μL, from Experiment 2) can be fed through channel 701; stock solution 2 (40 μL, from Experiment 2) and cells (100 cells / μL) can be fed through channel 702; and CB oil (200 μL, from Experiment 1) can be fed through channel 704. A collection of water-in-oil emulsions of droplets can be obtained in the collection wells. The emulsion of droplets can be maintained in the wells (with lids) for about 60 minutes. Subsequent solvent exchange can convert the oil phase into an aqueous phase (5 mM EDTA). The gel can be washed with phosphate-buffered saline (3×). Gelation can be observed. The gel captured by single cells can be observed under a microscope (e.g., Fig.19 The swelling ratio of the gel can be measured by comparing the size data between monodisperse droplets in the oil phase (100 min) and monodisperse beads in the water phase (5 min).

[0365] Experiment 5: Polymerization using AIBN as initiator

[0366] Copolymer poly(acrylamide) attached with click chemistry precursors can be synthesized by free radical solution polymerization as follows: acrylamide monomer (50 mmol, a mixture of acrylamide and its derivatives including click chemistry moieties), NaF (1.6 M) and 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044, 0.05 mmol) can be dissolved in water. The mixture can be bubbled with nitrogen for half an hour and subjected to 30°C to 40°C for 24 hours. After cooling to room temperature, the desired product can be obtained based on its solubility in aqueous solvents relative to organic solvents (hexane, EtOAc, EtOH, etc.).

[0367] Example 2: Synthesis of propargylated monomers and copolymers

[0368] The propargyl-containing monomer 2D can be synthesized as shown in Scheme 9. Propargyl alcohol is reacted with carbonyl-diimidazole to give the propargylating agent 2A, as shown. Monopropargylation of cystamine 2B with propargylating agent 2A provides monopropargylated cystamine 2C, which is further acylated on the free amine to provide disulfide-linked propargylated monomer 2D. Monomer 2D is a degradable alkyne-containing monomer.

[0369] Solution 9

[0370]

[0371] Copolymers with click chemistry precursors attached were synthesized by free radical solution polymerization as follows (see Schemes 10 and 11).

[0372] For the first copolymer, acrylamide monomer (50 mmol, a mixture of acrylamide and monomer 2D), NaF (1.6 M) and 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIBN, 0.05 mmol) were dissolved in water. The mixture was bubbled with nitrogen for 30 minutes and then subjected to 30°C to 40°C for 24 hours. After cooling to room temperature, the desired product was obtained based on its solubility in aqueous solvents relative to organic solvents (hexane, EtOAc, EtOH, etc.).

[0373] Solution 10

[0374]

[0375] For the second copolymer, poly(acrylamide) polymer and DMTMM were dissolved in water and the pH was corrected to 7.5 with NaOH. 3-azido-1-propylamine was added and the solution was stirred at room temperature in the dark for about 16 hours. The product was purified by dialysis and lyophilized to obtain the target copolymer as a white solid.

[0376] Solution 11

[0377]

[0378] Example 3: Single-cell DNA sequencing using cell beads generated by click chemistry

[0379] The synthesis of azide and alkyne polymers was performed as described in Example 2.

[0380] Preparation of copper(II) reagent in oil phase

[0381] 1) Suspension of copper (II) salt. At room temperature, about 1.375 mM Krytox-COOH and about 1.25 mM Cu(OAc) 2 Stir in HFE-7500 for about 48 hours.

[0382] 2) Prepare GB oil. GB oil is prepared by mixing about 2.50 mM bis-Krytox-ethylene glycol-polymer (Krytox-PEG-Krytox or BKEP or Formula I) and about 0.25 mM Krytox in a solvent HFE-7500 engineered oil. GB oil is pre-blended in batches.

[0383] 3) Prepare the oil phase solution CB oil. The suspension of copper (II) salt and GB oil are combined at a ratio of v / v=1:1, and the resulting mixture is stirred at room temperature for about 1 hour and filtered through a 0.22 μm PES filter to remove insoluble copper (II) salt. The filtrate is a CB oil including HFE-7500 containing 1.25 mM BKEP, about 1.375 mM Krytox-COOH and about 0.625 mM copper (II).

[0384] Preparation of aqueous solution

[0385] A stock solution including sodium ascorbate was prepared by mixing a polymer pair of an azide-containing (1.75 w / v%) and an alkyne-containing polymer (1.75 w / v%), F-108 (0.5 w / v%), magnetic particles (as indicated in Table 2), water containing THPTA (1.00 mM), sodium ascorbate (10.00 mM), and optionally DMSO (as indicated in Table 2) in water. A corresponding stock solution without sodium ascorbate was generated for each sample type for cell bead generation (see below).

[0386] Table 2

[0387]

[0388] Click chemistry and gelation in the presence of cells

[0389] To prepare an emulsion of discrete droplets, a method similar to Figure 7 The apparatus setup depicted in the apparatus setup is shown in Figure 2. Specifically, a 50 μL plate containing sodium ascorbate (60 μL) and BJ cells was used. A stock solution of is fed through the first channel (e.g., 701); a corresponding stock solution without sodium ascorbate (40 μL) is fed through the second channel (e.g., 702); and copper-free oil (270 μL) is fed through the third channel (e.g., 704). A collection of water-in-oil emulsions of droplets is obtained in the collection well. The emulsion of droplets is kept in the well (with lid) for about 15 minutes, with shaking at 1000 rpm. Then, CB oil is added to a final copper concentration of 0.9 mM. The emulsion of droplets is kept in the well for an additional 45 minutes, with shaking at 1000 rpm. Subsequent solvent exchange is used to convert the oil phase into an aqueous phase (5 mM EDTA). The gel is washed in phosphate-buffered saline (3×) to generate cell beads. Gelation is observed under the naked eye and microscope.

[0390] DNA Sequencing

[0391] The resulting cell beads were processed for DNA sequencing as described elsewhere herein. In short, the cell beads were separated into droplets of barcoded beads, lysed and the DNA from the cells was barcoded. The resulting barcoded DNA was separated and subjected to nucleic acid sequencing. The nucleic acid sequencing results for each sample were analyzed and compared with the target specifications for optimal cell bead performance. The results of this analysis are shown in Table 3.

[0392] Table 3

[0393]

[0394] Fig. 20 Sequencing results from one sample, AN400_0.06_DMSO, are shown, demonstrating high quality sequencing results using these conditions.

[0395] Example 4: Cell centering

[0396] The synthesis of azide and alkyne polymers was performed as described in Example 2.

[0397] Preparation of copper(II) reagent in oil phase

[0398] 1) Suspension of copper (II) salt. At room temperature, about 1.375 mM Krytox-COOH and about 1.25 mM Cu(OAc) 2 Stir in HFE-7500 for about 48 hours.

[0399] 2) Prepare GB oil. GB oil is prepared by mixing about 2.50 mM bis-Krytox-ethylene glycol-polymer (Krytox-PEG-Krytox or BKEP or Formula I) and about 0.25 mM Krytox in a solvent HFE-7500 engineered oil. GB oil is pre-blended in batches.

[0400] 3) Prepare the oil phase solution CB oil. The suspension of copper (II) salt and GB oil are combined at a ratio of v / v=1:1, and the resulting mixture is stirred at room temperature for about 1 hour and filtered through a 0.22 μm PES filter to remove insoluble copper (II) salt. The filtrate is a CB oil including HFE-7500 containing 1.25 mM BKEP, about 1.375 mM Krytox-COOH and about 0.625 mM copper (II).

[0401] Preparation of aqueous solution

[0402] A stock solution including sodium ascorbate was prepared by mixing a polymer pair containing an azide (1.75 w / v%) and an alkyne-containing polymer (1.75 w / v%), F-108 (0.5 w / v%), magnetic particles (0.12 w / v%), water containing THPTA (1.00 mM), and sodium ascorbate (150.00 mM) in water. A corresponding stock solution without sodium ascorbate was generated for each sample type for cell bead generation (see below).

[0403] Click chemistry and gelation in the presence of cells

[0404] To prepare an emulsion of discrete droplets, a method similar to Figure 7 . Specifically, a stock solution containing sodium ascorbate (60 μL) and peripheral blood mononuclear cells (PBMCs) obtained from a subject is fed through a first channel (e.g., 701); a corresponding stock solution without sodium ascorbate (40 μL) is fed through a second channel (e.g., 702); and copper-free oil (270 μL) is fed through a third channel (e.g., 704). A collection of water-in-oil emulsions of droplets is obtained in the collection wells. The emulsion of droplets is divided into two sets of wells and passed through Fig.21A The process was carried out according to the workflow shown. One set of emulsions was covered for about 15 minutes with shaking at 1000 rpm to promote cell centering. The second set was covered for about 15 minutes without shaking. Then, CB oil was added to a final copper concentration of 0.625 mM. The emulsion of droplets was kept in the well for an additional 45 minutes with shaking at 1000 rpm. Subsequent solvent exchange was used to convert the oil phase into an aqueous phase (5 mM EDTA). The gel was washed in phosphate buffered saline (3×) to generate cell beads. Gelation was observed with the naked eye and under a microscope.

[0405] Nuclear staining and imaging

[0406] The cell beads were Nuclear staining was performed and images were imaged using fluorescence microscopy. Fig. 21B(uncentered) and 21C (centered) show the imaging results. Fig. 22 Results of a cell centring assay are shown, indicating that emulsions subjected to a centring procedure (shaking) generated a greater number of cell beads, with cells closer to the centre of the beads, compared to emulsions not subjected to a centring procedure.

[0407] Example 5: Cellular bead generation parameters affect DNA degradation

[0408] The generated products include azide and alkyne polymers, λDNA and Fig.23 Emulsions of additional components as indicated in . Sodium ascorbate as provided at 150 mM. Each was generated using copper-free oil. The lambda DNA concentration of each was measured by quantitative PCR (qPCR). These results indicate that in the presence of paramagnetic particles (PMP) and sodium ascorbate (Na Asc), lambda DNA concentration decreases due to DNA degradation. Removing PMP from the conditions eliminates DNA degradation.

[0409] Example 6: DMSO addition and / or PMP selection affect DNA degradation

[0410] The resulting products include azide and alkyne polymers, DNA and Fig.24 Emulsions of additional components as indicated in . Each was generated using copper-containing oil. Sodium ascorbate was provided at 10 mM. Two different PMPs were tested; Sphero TM Carboxy magnetic particles (Sphero), which include an iron oxide coating surrounding a polystyrene core, and Dynabeads magnetic beads (Dyna), which include an iron oxide core surrounded by an outer polymer coating, each in different concentrations of DMSO, e.g. Fig.24 These results indicate that using Dynabeads and adding at least 5% DMSO can prevent DNA degradation.

[0411] Example 7: Generation of cell beads using CuAcAc

[0412] The use of copper(II) hexafluoroacetylacetonate (CuAcAc) as a copper source for click chemistry-mediated cell bead generation was tested under different parameters.

[0413] Fig.25A Results of cell bead generation using different sodium ascorbate (Na Asc) concentrations ranging from 10 mM to 200 mM are shown. Cell beads were generated as described herein using the following parameters: 1 mM THPTA, 1 mM CuAcAc, and 2.5 mM KmPEG oil. These results indicate that, under these conditions, 100 mM sodium ascorbate provides optimal cell bead generation.

[0414] Fig.25B The results of generating cell beads using different gelation times ranging from 0 minutes to overnight (ON) are shown. Cell beads were generated as described herein using the following parameters: 1 mM THPTA, 1 mM CuAcAc, 2.5 mM KmPEG oil, and 100 mM sodium ascorbate. These results indicate that under these conditions, a gelation time of 60 minutes is the shortest time required to achieve optimal cell bead generation.

[0415] Fig.25C The results of generating cell beads using different THPTA concentrations in the range of 0.5 mM to 8 mM are shown. Cell beads were generated as described herein using the following parameters: 1 mM CuAcAc, 2.5 mM KmPEG oil, and 20 mM sodium ascorbate. These results indicate that under these conditions, 5 mM THPTA provides the best cell bead generation.

[0416] Fig.25D Results of cell bead generation using different sodium ascorbate (Na Asc) concentrations ranging from 2.5 mM to 100 mM are shown. Cell beads were generated as described herein using the following parameters: 5 mM THPTA, 1 mM CuAcAc, and 2.5 mM KmPEG oil. These results indicate that, under these conditions, 50 mM sodium ascorbate provides optimal cell bead generation.

[0417] Fig.26A Results of cell bead generation using different CuAcAc concentrations in the range of 0.3125 mM to 3.75 mM are shown. Cell beads were generated as described herein using the following parameters: 5 mM THPTA, 2.5 mM KmPEG oil, and 20 mM sodium ascorbate. These results indicate that, under these conditions, 1 mM CuAcAc provides optimal cell bead generation.

[0418] Fig.26B The results of generating cell beads using different gelation times ranging from 0 minutes to overnight (ON) are shown. Cell beads were generated as described herein using the following parameters: 5mM THPTA, 1mM CuAcAc, 2.5mM KmPEG oil and 50mM sodium ascorbate. These results show that under these conditions, a gelation time of 15 minutes is the shortest time required to achieve optimal cell bead generation.

[0419] Table 4 shows the optimal parameters identified for generating cell beads using CuAcAc, requiring only 15 min of gelation time.

[0420] Table 4

[0421] Components Final concentration Azide polymer blend 1.75w / v% Alkyne polymer mixture 1.75w / v% F-108 0.50w / v% Magnetic particles (PMP) 0.12w / v% Ligand (THPTA) 5.00mM Reducing agent (Na Asc). 50.00mM Additive (DMSO) 5v / v%

[0422] Example 8: Using Cu 2 OAc Producing Cell Beads

[0423] The use of copper acetate (Cu 2 OAc) as a copper source for click chemistry-mediated cell bead generation.

[0424] Fig.27A Results of cell bead generation using different THPTA concentrations ranging from 0.05 mM to 8 mM are shown. Cell beads were generated as described herein using the following parameters: 5 mM sodium ascorbate, 0.625 mM Cu 2 OAc and 2.5 mM KmPEG oil. These results indicate that 1 mM THPTA provided optimal cell bead generation under these conditions. No gelation was observed for 0.05 mM, 5 mM, and 8 mM THPA concentrations.

[0425] Fig.27B Results are shown for cell beads generated using different sodium ascorbate (Na Asc) concentrations ranging from 2.5 mM to 100 mM. Cell beads were generated as described herein using the following parameters: 1 mM THPTA, 0.625 mM Cu 2 OAc and 2.5 mM KmPEG oil. These results indicate that 50 mM sodium ascorbate provides optimal cell bead generation under these conditions.

[0426] Fig.27C Results are shown for cell beads generated using different sodium ascorbate (Na Asc) concentrations ranging from 2.5 mM to 100 mM. Cell beads were generated as described herein using the following parameters: 1 mM THPTA, 1 mM Cu 2 OAc and 2.5 mM KmPEG oil. These results indicate that 20 mM sodium ascorbate provides optimal cell bead generation under these conditions.

[0427] Fig.27D Results are shown for cell beads generated using different sodium ascorbate (Na Asc) concentrations ranging from 2.5 mM to 100 mM. Cell beads were generated as described herein using the following parameters: 1 mM THPTA, 2 mM Cu 2 OAc and 2.5 mM KmPEG oil. These results indicate that 20 mM sodium ascorbate provides optimal cell bead generation under these conditions.

[0428] Fig.28A Results are shown for cell beads generated using THPTA concentrations ranging from 0.025 mM to 5 mM. Cell beads were generated as described herein using the following parameters: 2 mM Cu2 OAc, 20 mM sodium ascorbate, and 2.5 mM KmPEG oil. These results indicate that 1 mM THPTA provides optimal cell bead generation under these conditions.

[0429] Fig.28B Results are shown for cell beads generated using different gelation times ranging from 0 minutes to overnight (ON). Cell beads were generated as described herein using the following parameters: 1 mM THPTA, 2 mM Cu 2 OAc, 2.5 mM KmPEG oil, and 20 mM sodium ascorbate. These results indicate that under these conditions, a gelation time of 30 minutes is the minimum time required to achieve optimal cell bead generation.

[0430] Table 5 shows the identification of Cu 2 OAc, only 30 min of gelation time was required to generate optimal parameters for cell beads.

[0431] Table 5

[0432] Components Final concentration Azide polymer blend 1.75w / v% Alkyne polymer mixture 1.75w / v% F-108 0.50w / v% Magnetic particles (PMP) 0.12w / v% Ligand (THPTA) 1.00mM Reducing agent (Na Asc). 20.00mM Additive (DMSO) 5v / v%

[0433] Example 9: Generation of Copper Nanoparticle / Cell Complexes

[0434] Copper nanoparticles (CuNP) were obtained from US Research Nanomaterials Inc. 10 mg CuNP was suspended in 1 mL complete cell culture medium. CuNP was ultrasonically treated for 2 minutes and then vortexed for 10 minutes. 10 mL complete cell culture medium was added to the CuNP suspension and centrifuged for 5 minutes at 150 g to remove large particles. The supernatant was collected and centrifuged for 5 minutes at 1000 g. The supernatant was discarded and the precipitate was resuspended in 1 mL complete cell culture medium. The resulting CuNP suspension was ultrasonically treated for 30 minutes in a bath sonicator.

[0435] Next, cells were cultured at 10 7 The cells were resuspended in complete cell culture medium. 1 mL of CuNP dispersion was added to the cell suspension and incubated at 4 ° C for 15 minutes. Then, the mixture was centrifuged at 50 g for 5 minutes at 4 ° C, gently mixed, and centrifuged again using the same conditions. The cell pellet was washed twice with 10 mL of complete cell culture medium, centrifuged at 20 g at 4 ° C, and the supernatant was removed after each wash. Finally, the CuNP / cell mixture was resuspended in 500 μL of complete culture medium to generate cell beads.

[0436] Example 10: Use of copper nanoparticles for cell bead generation

[0437] The CuNP / cell mixture is separated into droplets as described herein along with the polymer for cell bead generation. The polymer in the droplets including the cell / CuNP complex is cross-linked using CuNP as a catalyst by click chemistry to generate cell beads. The polymer in the droplets not including the cell / CuNP complex is not cross-linked. A cell bead population is generated.

[0438] Example 11: Generation of low copper concentration cell beads using pyridylmethyl polymers

[0439] This example demonstrates how to generate cell beads (CBs) from an emulsion of discrete droplets at low copper concentrations using a polymer with copper-chelating azidopyridylmethyl functional groups. Fig.29 As schematically depicted and described elsewhere herein, a linker modified with an azido-pyridylmethyl functional group can undergo a copper-catalyzed click reaction with an alkyne-modified linker to form a crosslink comprising a 1,2,3-triazole click chemical bond. Further, the ability of the azido-pyridylmethyl functional group to chelate copper ions effectively increases the copper concentration at the site of the copper-catalyzed click reaction. This increase in the effective copper concentration at the reaction site allows the total copper concentration in the reaction to be reduced without losing crosslinking efficiency. The reduction in total copper concentration during cell bead generation significantly improves biocompatibility, such as degradation of RNA components.

[0440] Two sets of CBs were generated from emulsions of discrete droplets according to the method described in Example 3. One set of CBs contained polymers with azido-pyridylmethyl functional groups, while the other set had polymers with standard azide functional groups. The generation of both sets of CBs was performed by copper-catalyzed click chemistry over a copper concentration range of 0.0625 mM to 0.625 mM. The size of the CBs generated under different copper concentration conditions was determined by microscopy.

[0441] like Fig.31A As shown, significant differences are observed in the CB formed using pyridylmethyl polymers and the CB formed using standard non-pyridylmethyl polymers. When Cu concentration is reduced by half (from 0.625mM to 0.3125mM), the swelling ratio (SR) of pyridylmethyl polymer CB shows no change. In addition, using the Cu concentration of 0.0625mM in 37C oil, only pyridylmethyl polymer CB can be generated in 30 minutes, and SR is only slightly increased from 0.70 to 0.81. By contrast, non-pyridylmethyl polymer CB cannot be generated under the Cu concentration of 0.0625mM, and even Cu concentration is reduced to 0.3125mM from 0.625mM causing the CB swelling ratio to significantly increase from 0.70 to 1.07.

[0442] Further experiments were performed in which CBs were generated from emulsions of droplets containing GM12878 cells dispersed in a polymer mixture containing polymers with azido-picolyl functional groups at low Cu concentrations (0.15 mM or 0.20 mM) or polymers with azido-alkyl (i.e., non-picolyl) functional groups at higher Cu concentrations (0.625 mM). For droplets containing either picolyl or non-picolyl polymers, gelation within the droplets was performed at room temperature for 45 minutes under the following conditions: (a) picolyl polymers: 0.15 mM Cu, 8 mM sodium ascorbate; or 0.20 mM Cu, 10 mM sodium ascorbate; (b) non-picolyl polymers: 0.625 mM Cu, 10 mM sodium ascorbate. After gelation, the emulsions were broken and the resulting CBs were washed twice in PBS and then packaged by centrifugation. Equal amounts of different sets of CBs (depending on the copper concentration) were added to the 3'RT mix, and a standard "single cell 3'v2" protocol was performed to generate cDNA products (10x Genomics, Pleasanton, CA, USA). The DTT in the 3'RT mix was used to degrade the disulfide crosslinks of the hydrogel matrix, thereby dissolving the CBs.

[0443] As shown by the results depicted in Figure 31B, in CBs formed by click chemistry cross-linking polymers with azido-picolyl functional groups and lower copper concentrations (0.15 mM or 0.2 mM), about 95% of genes and 94% of unique molecular identifiers (UMIs) were detected relative to the cell control. In contrast, in CBs formed using non-picolyl polymers and higher copper concentrations (0.625 mM), only 45% of genes and only 33% of UMIs were detected relative to the cell control.

[0444] In summary, the ability to use significantly lower Cu concentrations during gelation of discrete droplets to form CBs can be achieved by incorporating polymers with azido-pyridylmethyl functional groups. The use of lower copper concentrations in forming CBs resulted in significantly reduced RNA degradation and significantly improved gene detection.

[0445] Example 12: Chemically degraded cell beads with urethane bonds

[0446] This example demonstrates how CBs generated from emulsions of discrete droplets with labile urethane (rather than disulfide) bonds can be selectively degraded using diethyltriamine (DETA) and heat.

[0447] Emulsions of discrete droplets are generated from polymers modified with linkers containing either azide or alkyne groups that enable CuAAC click chemistry. Fig.32 As shown in the depicted scheme, the linker with an alkyne group includes a propargyl-carbamate moiety, which undergoes a copper-catalyzed click chemistry cross-linking reaction with an azide linker-modified polymer to form a gel matrix. The crosslinks that form the gel matrix include 1,2,3-triazole moieties but do not contain disulfide bonds. Therefore, they are not easily degraded by DTT treatment. Alternatively, the crosslinks can be degraded by treatment with a polyamine (e.g., DETA) and heat (e.g., 60°C), which is used to cleave the carbamate groups such as Fig.32 As shown.

[0448] CBs were generated from droplet emulsions as described in Example 3, except that the linker included a propargyl-carbamate moiety (and no disulfide bonds), such as Fig.32 As shown. The degradation characteristics of the resulting CB including carbamate bonds in the presence of DETA and heat were analyzed. Degradation was monitored using an optical microscope. 30 μL of carbamate CB was exposed to 200 μL of PBS solution containing 10% DETA at 60°C and compared to carbamate CB exposed to a control PBS solution without DETA. The carbamate CB was completely degraded after 15 minutes in 10% DETA at 60°C.

[0449] For further comparison, gel beads (GB) without carbamate bonds (10x Genomics, Pleasanton, CA, USA) were also treated with 10% DETA and heat, and degradation was monitored under a microscope. Fig.33 As shown in the data graph depicted, upon treatment with 10% DETA and 60°C heat, the CB with urethane-containing crosslinks swelled from -60 μm to -110 μm within the first 10 minutes of treatment and was completely dissolved after 15 minutes of treatment. In contrast, the GB with no urethane-containing crosslinks did not change significantly after 15 minutes, indicating no degradation in the presence of 10% DETA and heating to 60°C.

[0450] Notwithstanding the appended claims, the disclosure set forth herein is also defined by the following clauses, which may be beneficial alone or in combination with one or more other causes or embodiments. Without limiting the foregoing description, certain non-limiting clauses of the disclosure are provided as numbered below, wherein each of the individually numbered clauses may be used or combined with any of the preceding or following clauses. Therefore, this is intended to provide support for all such combinations and is not necessarily limited to the specific combinations expressly provided below:

[0451] 1. A composition comprising: (a) a bioparticle and / or a macromolecular component; and (b) two or more cross-linked polymers, wherein the cross-links are formed by click chemistry.

[0452] 2. The composition according to clause 1, wherein the cross-linked polymer is a gel.

[0453] 3. The composition according to clause 1, wherein the cross-linked polymer encapsulates the biological particles and / or macromolecular components.

[0454] 4. The composition of clause 1, wherein the crosslink comprises a picolyl moiety.

[0455] 5. The composition according to clause 1, wherein the click chemistry is copper-catalyzed.

[0456] 6. The composition of clause 5, wherein the copper-catalyzed click chemistry comprises a copper concentration selected from about 0.3 mM or less, about 0.2 mM or less, and about 0.15 mM or less.

[0457] 7. The composition according to clause 1, wherein the click chemistry is copper-free.

[0458] 8. A composition according to claim 7, wherein the copper-free click chemistry is selected from: (a) strain-promoted azide / dibenzocyclooctyne-amine (DBCO) click chemistry; (b) inverse electron demand Diels-Alder (IED-DA) tetrazine / trans-cyclooctene (TCO) click chemistry; (c) inverse electron demand Diels-Alder (IED-DA) tetrazine / norbornene click chemistry; (d) Diels-Alder maleimide / furan click chemistry; (e) Staudinger ligation; and (f) nitrile oxide / norbornene cycloaddition click chemistry.

[0459] 9. The composition of clause 8, wherein the crosslink comprises a 1,2,3-triazole moiety.

[0460] 10. The composition of clause 8, wherein the crosslink comprises a dihydropyridazine moiety.

[0461] 11. The composition of clause 1, wherein the crosslinks comprise labile bonds.

[0462] 12. The composition according to clause 11, wherein the labile bond is selected from a chemically labile bond, a thermally labile bond, an enzymatically labile bond, a photolabile bond, or a combination thereof.

[0463] 13. The composition according to clause 11, wherein the labile bond is selected from a disulfide bond, a carbamate bond, a peptide bond or a combination thereof.

[0464] 14. The composition of clause 1, wherein the composition further comprises at least one agent that is linked to at least one of the polymers by click chemistry.

[0465] 15. The composition according to clause 14, wherein the at least one agent is an oligonucleotide.

[0466] 16. A composition according to clause 15, wherein the oligonucleotide comprises a poly-T sequence.

[0467] 17. A method of forming a gel, comprising: combining in partitions under click chemistry reaction conditions (i) biological particles and / or macromolecular components, and (ii) two or more polymers configured to be cross-linked by click chemistry.

[0468] 18. The method of clause 17, wherein one of the polymers configured to be cross-linked comprises a picolyl moiety.

[0469] 19. The method according to clause 17, wherein the click chemistry reaction conditions include a copper concentration selected from about 0.3 mM or less, about 0.2 mM or less, and about 0.15 mM or less.

[0470] 20. The method of clause 17, wherein the click chemistry reaction conditions are copper-free.

[0471] 21. A method according to claim 20, wherein the two or more polymers are configured to be cross-linked via a copper-free click chemistry selected from the following: (a) strain-promoted azide / dibenzocyclooctyne-amine (DBCO) click chemistry; (b) inverse electron demand Diels-Alder (IED-DA) tetrazine / trans-cyclooctene (TCO) click chemistry; (c) inverse electron demand Diels-Alder (IED-DA) tetrazine / norbornene click chemistry; (d) Diels-Alder maleimide / furan click chemistry; (e) Staudinger ligation; and (f) nitrile oxide / norbornene cycloaddition click chemistry.

[0472] 22. The method of clause 20, wherein the click chemistry bond comprises a 1,2,3-triazole moiety.

[0473] 23. The method of clause 20, wherein the click chemistry bond comprises a dihydropyridazine moiety.

[0474] 24. The method of clause 17, wherein the formed crosslinks comprise labile bonds.

[0475] 25. The method according to clause 24, wherein the labile bond is a chemically labile bond, a thermally labile bond, an enzymatically labile bond or a photolabile bond.

[0476] 26. The method according to clause 24, wherein the labile bond is a disulfide bond, a carbamate bond or a peptide bond.

[0477] 27. The method of clause 17, wherein the partitions are holes.

[0478] 28. The method of clause 17, wherein the partitions are discrete droplets in an emulsion.

[0479] 29. A composition comprising: (a) a biological sample; and (b) a gel of two or more cross-linked polymers, wherein the cross-links are formed by click chemistry using a linker comprising a copper chelating group.

[0480] 30. The composition of clause 29, wherein the crosslink comprises a picolyl moiety.

[0481] 31. A composition according to any one of clauses 29 to 30, wherein the copper chelating group is a picolyl group; optionally wherein the picolyl group is an azidopicolyl group.

[0482] 32. A composition according to any one of clauses 29 to 31, wherein the click chemistry is copper-catalyzed; optionally wherein the copper-catalyzed click chemistry comprises a copper concentration selected from about 0.3 mM or less, about 0.2 mM or less, and about 0.15 mM or less.

[0483] 33. A composition according to any of clauses 29 to 32, wherein one polymer of the two or more polymers comprises at least one member selected from the group consisting of polyolefins, olefin copolymers, acrylics, vinyl polymers, polyesters, polyamides, polyimides, formaldehyde resins, polyurethanes, ether polymers, celluloses, thermoplastic elastomers and thermoplastic polyurethanes.

[0484] 34. A composition according to any one of clauses 29 to 33, wherein the gel is a hydrogel.

[0485] 35. A composition according to any one of clauses 29 to 34, wherein the cross-linked polymer is distributed throughout the gel.

[0486] 36. A composition according to any one of clauses 29 to 35, wherein the cross-linked polymer encapsulates the biological sample; optionally wherein the biological sample comprises a cell, a cell nucleus or one or more components derived from the cell.

[0487] 37. The composition of any one of clauses 29 to 36, wherein one polymer of the two or more polymers is polyacrylamide.

[0488] 38. The composition according to any one of clauses 29 to 37, wherein the composition further comprises at least one agent that is linked to at least one of the polymers by click chemistry.

[0489] 39. A composition comprising: (a) a biological sample; and (b) a gel of two or more cross-linked polymers, wherein the cross-links are formed by copper-free click chemistry, wherein the copper-free click chemistry is selected from: (i) strain-promoted azide / dibenzocyclooctyne-amine (DBCO) click chemistry; (ii) inverse electron demand Diels-Alder (IED-DA) tetrazine / trans-cyclooctene (TCO) click chemistry; (iii) inverse electron demand Diels-Alder (IED-DA) tetrazine / norbornene click chemistry; (iv) Diels-Alder maleimide / furan click chemistry; and (v) Staudinger ligation; and (iv) nitrile oxide / norbornene cycloaddition click chemistry.

[0490] 40. The composition of clause 39, wherein the crosslink comprises a 1,2,3-triazole moiety.

[0491] 41. The composition of clause 39, wherein the crosslink comprises a dihydropyridazine moiety.

[0492] 42. A composition according to any of clauses 39 to 41, wherein one polymer of the two or more cross-linked polymers comprises at least one member selected from the group consisting of polyolefins, olefin copolymers, acrylics, vinyl polymers, polyesters, polyamides, polyimides, formaldehyde resins, polyurethanes, ether polymers, celluloses, thermoplastic elastomers and thermoplastic polyurethanes.

[0493] 43. A composition according to any one of clauses 39 to 42, wherein the gel is a hydrogel.

[0494] 44. A composition according to any one of clauses 39 to 43, wherein the cross-linked polymer is distributed throughout the gel.

[0495] 45. A composition according to any one of clauses 39 to 44, wherein the cross-linked polymer encapsulates the biological sample; optionally wherein the biological sample comprises a cell, a cell nucleus or one or more components derived from the cell.

[0496] 46. ​​The composition of any one of clauses 39 to 45, wherein one polymer of the two or more cross-linked polymers is polyacrylamide.

[0497] 47. A composition comprising: (a) a biological sample; and (b) a gel of two or more cross-linked polymers, and wherein the cross-links comprise labile bonds.

[0498] 48. The composition according to clause 47, wherein the labile bond is selected from a disulfide bond, a carbamate bond, a peptide bond or a combination thereof.

[0499] 49. The composition according to any one of clauses 47 to 48, wherein the composition further comprises at least one agent that is linked to at least one of the polymers by click chemistry.

[0500] 50. The composition according to clause 49, wherein the at least one agent is an oligonucleotide.

[0501] 51. A composition according to clause 50, wherein the oligonucleotide comprises a poly-T sequence.

[0502] 52. A composition according to any one of clauses 47 to 51 wherein the gel is a hydrogel.

[0503] 53. A composition according to any one of clauses 47 to 52, wherein the cross-linked polymer is distributed throughout the gel.

[0504] 54. A composition according to any one of clauses 47 to 53, wherein the cross-linked polymer encapsulates the biological sample; optionally wherein the biological sample comprises a cell, a cell nucleus or one or more components derived from the cell.

[0505] Although preferred embodiments of the present invention have been shown and described herein, it is obvious to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided in the specification. Although the present invention has been described with reference to the above description, the description and illustration of the embodiments herein are not intended to be understood in a limiting sense. Without departing from the present invention, many variations, changes and substitutions will now occur to those skilled in the art. In addition, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations or relative proportions depending on various conditions and variables set forth herein. It should be understood that different alternatives to the embodiments of the present invention described herein may be used to practice the present invention. It is therefore contemplated that the present invention should also encompass any such substitutions, modifications, variations or equivalents. The following claims are intended to define the scope of the present invention and thus cover methods and structures within the scope of these claims and their equivalents.

Claims

1. A partition comprising a composition, wherein the composition comprises: (a) a biological sample, wherein the biological sample comprises a cell, one or more components derived from a cell, or a virus; as well as (b) a gel of two or more cross-linked polymers, wherein the two or more cross-linked polymers include copper chelating groups and are configured to encapsulate the biological sample; The partition is a droplet in a plurality of droplets or a well in a plurality of wells.

2. The partition of claim 1, wherein the two or more cross-linked polymers comprise picolyl moieties.

3. The partition of claim 1, wherein the copper chelating group is a pyridylmethyl group.

4. The partition of claim 1, wherein the gel comprises a copper concentration of 0.3 mM or less.

5. The partition of claim 1, wherein one of the two or more cross-linked polymers comprises at least one member selected from the group consisting of polyolefins, acrylics, polyesters, polyamides, polyimides, formaldehyde resins, polyurethanes, ether polymers, celluloses, and thermoplastic elastomers.

6. The partition of claim 1, wherein one of the two or more cross-linked polymers comprises an olefin copolymer.

7. The partition of claim 1, wherein one of the two or more cross-linked polymers comprises a vinyl polymer.

8. The partition of claim 1, wherein one of the two or more cross-linked polymers comprises thermoplastic polyurethane.

9. The partition of claim 1, wherein the gel is a hydrogel.

10. The partition of claim 1, wherein the biological sample comprises a cell nucleus.

11. The partition of claim 1, wherein the biological sample comprises cells.

12. The partition of claim 1, wherein one of the two or more cross-linked polymers is polyacrylamide.

13. The partition of claim 1, further comprising at least one agent attached to at least one of the two or more cross-linked polymers through a thiazole group.

14. The partition of claim 13, wherein the at least one reagent comprises an oligonucleotide.

15. The partition of claim 14, wherein the oligonucleotide comprises a poly-T sequence.

16. The partition of claim 1, wherein the two or more cross-linked polymers comprise a labile bond selected from a disulfide bond, a carbamate bond, a peptide bond, or a combination thereof.

Images