Method and hydrogel composition for partitioning biological sample

Abstract

Methods of making and forming partitions in nanovials are provided. Disclosed is a method in which an oligonucleotide is linked to a cellular nucleic acid in a hollow nanovial, the method comprising loading a cell into an opening in a hollow nanovial; one or more hydrogel beads are inserted into the opening, thereby blocking the opening and preventing diffusion of the cells from the opening.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 347,419, filed on May 31, 2022, which is incorporated by reference for all purposes. Background Art

[0003] Partitioning reagents has been found to be useful for many molecular reactions. For example, droplet-based single-cell sequencing allows for high-resolution gene sequencing.

[0004] In single-cell next-generation sequencing (NGS) sample preparation, complex microfluidics are required to deliver cells and beads into barcoded partitions. This complexity increases the cost of the solution because it usually requires a microfluidic chip as well as a microfluidic controller. In the case where the partitions are droplets, the chip generates the droplets and the instrument performs fluidic pressure control to deliver the partitions.

[0005] Furthermore, since partition creation is typically performed as a continuous operation, there is an upper limit to the number of droplets that are generated. This is because the time required to generate an increased number of partitions increases proportionally. Additionally, the extra time required means that cells can sit in buffer upstream of partition generation, so that the biology may begin to be affected, which will affect the biological data and results.

[0006] After the barcoded partitions are formed, it may be necessary to image the contents of the partitions to analyze, for example, secreted products produced by the cells, and then sort the cells of interest based on the imaging results. This is difficult in the case of water-in-oil droplets because the droplets are incompatible with cell sorters and microwells that are physically fixed in space.

[0007] Finally, typical barcoded partitions may not allow for buffer exchange because the partitions are fluidically separated from each other. This prevents multi-step biochemical reactions, some of which are required for barcoding complex cellular substrates such as methylated DNA. Summary of the invention

[0008] In some embodiments, a method for connecting an oligonucleotide to a cellular nucleic acid in a hollow nanovial or microwell is provided. In some embodiments, the method comprises:

[0009] The cells are loaded into the openings in the hollow nanovials or microwells;

[0010] inserting one or more hydrogel beads into the opening, thereby blocking the opening and preventing the cells from diffusing from the opening;

[0011] allowing the cells to lyse in the hollow nanovial or microwell;

[0012] One or more oligonucleotides are linked to the nucleic acids produced by the lysed cells, thereby barcoding the nucleic acids, wherein the oligonucleotides are present in the hollow nanovial or microwell prior to the loading or are introduced into the hollow nanovial or microwell via the hydrogel beads.

[0013] In some embodiments, the blocking comprises inducing swelling of one or more hydrogel beads inserted into the nanovial or microwell. In some embodiments, the blocking comprises inducing swelling of the nanovial.

[0014] In some embodiments, the majority of the nanovials or microwells contain one or zero cells. In some embodiments, to achieve one or zero cell loading, the average number of cells as a fraction of λ or alternatively nanovials or microwells is about 0.05 to 0.1 cells / nanovial or microwell. In some embodiments, to achieve one or zero cell loading, the average number of cells as a fraction of λ or alternatively nanovials or microwells is about 0.01 to 0.4 cells / nanovial or microwell.

[0015] In some embodiments, the oligonucleotide is attached to the hydrogel bead and is optionally released from the hydrogel bead after sealing. In some embodiments, the oligonucleotide is attached to the hydrogel bead and is optionally released from the hydrogel bead before the attachment. In some embodiments, the oligonucleotide has a barcode sequence, wherein a separate hydrogel bead comprises a cloned copy of the oligonucleotide, and wherein the barcode sequence attached to the separate hydrogel bead is unique, such that the barcode distinguishes the hydrogel bead from other hydrogel beads.

[0016] In some embodiments, the oligonucleotide has a barcode sequence, wherein an individual nanovial or microwell contains a clonal copy of the oligonucleotide, and wherein the sequenced barcode associated with an individual nanovial or microwell is unique such that the barcode distinguishes the nanovial or microwell from other nanovials or microwells.

[0017] In some embodiments, the method further comprises extracting the barcoded nucleic acid from the nanovial or microwell, and combining the barcoded nucleic acids from different nanovials or microwells into a bulk solution, and performing nucleotide sequencing on the barcoded nucleic acids from the bulk solution.

[0018] In some embodiments, the lysis comprises diffusing one or more lysis reagents into the cells in the nanovial or microwell. In some embodiments, the lysis reagent is removed or inactivated from the nanovial or microwell prior to the connection.

[0019] In some embodiments, the lysis comprises diffusing a protein denaturing and / or degrading agent into the cell in the nanovial or microwell. In some embodiments, the lysis agent is removed or inactivated from the nanovial or microwell prior to the connection.

[0020] In some embodiments, the ligating comprises ligating the oligonucleotide to the nucleic acid, and optionally performing a template-dependent extension of the oligonucleotide using the nucleic acid as a template.

[0021] In some embodiments, the ligation comprises diffusing one or more ligation reagents into a sealed nanovial or microwell containing the lysed cells. In some embodiments, the nucleic acid is DNA and the ligation reagent comprises a DNA polymerase or a ligase. In some embodiments, the nucleic acid is RNA and the ligation reagent comprises a reverse transcriptase.

[0022] In some embodiments, said ligating comprises ligating said oligonucleotide to said nucleic acid by a click chemistry reaction.

[0023] In some embodiments, the hollow nanovial comprises dextran and polyethylene glycol (PEG).

[0024] In some embodiments, the hydrogel beads comprise polyacrylamide, polystyrene, silica, polymethyl methacrylate, alginate, polyethylene glycol (PEG), nylon, or agarose.

[0025] In some embodiments, the hydrogel beads comprise polyacrylamide cross-linked with bisacryloylcystamine.In some embodiments, swelling is induced in the hydrogel beads by contacting the hydrogel beads with a disulfide bond reducing agent.

[0026] In some embodiments, the hydrogel beads comprise polyacrylamide formed by polymerizing acrylamide and a photocleavable crosslinker. In some embodiments, the photocleavable crosslinker is o-nitrobenzyl (o-NB) diacrylate. In some embodiments, swelling is induced in the hydrogel beads by contact light cleavage of the photocleavable crosslinker.

[0027] In certain embodiments, a variety of hollow nano vials or micropores are provided, which include blocked openings, and the blocked openings prevent cells and released cell products from spreading. In certain embodiments, at least some of the hollow nano vials or micropores include one or more cells, and the opening is blocked by the hydrogel beads that close the opening. In certain embodiments, the blocked opening is formed by introducing one or more hydrogel beads into the opening and then inducing the hydrogel beads to swell so that the hydrogel beads block the opening. In certain embodiments, the hollow nano vials include dextran and polyethylene glycol (PEG). In certain embodiments, the hydrogel beads include polyacrylamide, polystyrene, silica, polymethyl methacrylate, alginate, polyethylene glycol (PEG), nylon or agarose. In certain embodiments, most of the multiple hollow nano vials or micropores contain one or zero cells. In some embodiments, a separate hydrogel bead comprises a cloned copy of an oligonucleotide, and the oligonucleotide has a barcode sequence, and wherein the barcode sequence attached to the separate hydrogel bead is unique, such that the barcode distinguishes the hydrogel bead from other hydrogel beads. In some embodiments, the barcode sequence is attached to the separate hydrogel bead by click chemistry. In some embodiments, the oligonucleotide has a barcode sequence, wherein a separate nanovial or microwell comprises a cloned copy of an oligonucleotide, and the oligonucleotide has a barcode sequence, and wherein the sequenced barcode attached to the separate nanovial or microwell is unique, such that the barcode distinguishes the nanovial or microwell from other nanovials or microwells.

[0028] In some embodiments, the oligonucleotide has a barcode sequence, wherein a separate nanovial contains a cloned copy of the oligonucleotide, and the oligonucleotide has a barcode sequence, and wherein the barcode sequence attached to the separate nanovial is unique, such that the barcode distinguishes the nanovial from other nanovials. In some embodiments, the barcode sequence is attached to the separate nanovial by click chemistry.

[0029] In some embodiments, the hydrogel beads comprise polyacrylamide cross-linked with bisacryloylcystamine.

[0030] In some embodiments, the hydrogel beads comprise polyacrylamide formed by polymerizing acrylamide and a photocleavable cross-linker. In some embodiments, the photocleavable cross-linker is o-nitrobenzyl (o-NB) diacrylate.

[0031] A method for detecting cellular proteins in a hollow nanovial or microwell is also provided. In some embodiments, the method comprises:

[0032] The cells are loaded into the openings in the hollow nanovials or microwells;

[0033] inserting one or more hydrogel beads into the opening, thereby blocking the opening and preventing the cells from diffusing from the opening; and

[0034] Detection of secreted cellular proteins in blocked nanovials.

[0035] In some embodiments, the nanovial or microwell comprises a binding agent (which may be, but is not limited to, an antibody, biotin, or streptavidin) that specifically binds to one or more secreted cellular proteins, and the secreted cellular proteins bind to the binding agent, and a second binding agent (which may be directly or indirectly labeled) specific for the secreted cellular proteins diffuses into the blocked nanovial or microwell to specifically detect the secreted cellular proteins bound in the nanovial or microwell. For example, the second binding agent (e.g., an antibody or other binding agent) is typically conjugated or otherwise associated with a detectable label. The association may be direct, such as a covalent bond, or indirect, such as using a second binding agent, a chelating agent, or a linker.

[0036] definition

[0037] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described below are those well known and commonly used in the art. Standard techniques are used for nucleic acid and peptide synthesis. Techniques and procedures are generally performed according to conventional methods in the art and various general references (generally referring to, Sambrook et al. "Molecular Cloning: A Laboratory Manual (MOLECULAR CLONING: ALABORATORY MANUAL)", 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, the document is incorporated herein by reference), and the conventional methods and various general references are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry and organic synthesis described below are those well known and commonly used in the art.

[0038] As used herein, the terms "a," "an," or "the" include not only aspects having one member, but also aspects having more than one member. For example, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a bead" includes a plurality of such beads, and reference to "the sequence" includes reference to one or more sequences known to those of skill in the art, etc.

[0039] The term "amplification reaction" refers to any in vitro means of amplifying copies of a target sequence of a nucleic acid in a linear or exponential manner. Such methods include, but are not limited to, two-primer methods, such as polymerase chain reaction (PCR); ligase methods, such as DNA ligase chain reaction (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., ed., 1990)) (LCR); QBeta RNA replicase and RNA transcription-based amplification reactions (e.g., amplification involving RNA polymerization initiated by T7, T3 or SP6), such as transcription amplification system (TAS), nucleic acid sequence-based amplification (NASBA) and self-directed sequence replication (3SR); isothermal amplification reactions (e.g., single primer isothermal amplification (SPIA)); and other techniques known to those skilled in the art.

[0040] "Amplification" refers to a step in which a solution is placed under conditions sufficient to allow polynucleotide amplification, with all components of the reaction intact. The components of the amplification reaction include, for example, primers, polynucleotide templates, polymerases, nucleotides, etc. The term "amplification" generally refers to an "exponential" increase in a target nucleic acid. However, as used herein, "amplification" may also refer to a linear increase in the number of a selected target sequence of a nucleic acid, such as obtained by cycle sequencing or linear amplification. In an exemplary embodiment, amplification refers to a PCR amplification performed using a first amplification primer and a second amplification primer.

[0041] "Primer" refers to a polynucleotide sequence that hybridizes with a sequence on a target nucleic acid and serves as a starting point for nucleic acid synthesis. Primers can have a variety of lengths, and the length is generally less than 50 nucleotides, for example, 12-30 nucleotides in length. The length and sequence of primers used for PCR can be designed based on principles known to those skilled in the art, see, for example, Innis et al., supra. Primers can be chimeras of DNA, RNA, or a DNA portion and an RNA portion. In some cases, primers can include one or more modified or non-natural nucleotide bases. In some cases, primers are labeled.

[0042] Nucleic acid or a part thereof "hybridizes" with another nucleic acid under certain conditions so that non-specific hybridization is minimized at a limited temperature in a physiological buffer (e.g., pH 6-9, 25-150mM chloride salt). In some cases, nucleic acid or a part thereof hybridizes with a conserved sequence common in a group of target nucleic acids. In some cases, if there are at least about 6, 8, 10, 12, 14, 16 or 18 continuous complementary nucleotides, including "universal" nucleotides complementary to more than one nucleotide partner, primers or a part thereof can hybridize with primer binding sites. Alternatively, if there are 0 or less than 2 or 3 complementary mispairings on at least about 12, 14, 16, 18 or 20 continuous nucleotides, primers or a part thereof can hybridize with primer binding sites. In certain embodiments, the limited temperature at which specific hybridization occurs is room temperature. In certain embodiments, the limited temperature at which specific hybridization occurs is higher than room temperature. In some embodiments, the defined temperature for specific hybridization to occur is at least about 37° C., 40° C., 42° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., or 80° C. In some embodiments, the defined temperature for specific hybridization to occur is 37° C., 40° C., 42° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., or 80° C.

[0043] "Template" refers to a polynucleotide sequence comprising a polynucleotide to be amplified, adjacent to a primer hybridization site, or flanking a pair of primer hybridization sites. Thus, a "target template" comprises a target polynucleotide sequence adjacent to at least one hybridization site for a primer. In some cases, a "target template" comprises a target polynucleotide sequence flanked by hybridization sites for a "forward" primer and a "reverse" primer.

[0044] As used herein, "nucleic acid" means DNA, RNA, single-stranded, double-stranded or more highly aggregated hybridization motifs and any chemical modifications thereof. Modifications include, but are not limited to, providing modifications of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interactions, attachment points and functions of nucleic acid ligand bases or entire nucleic acid ligands. Such modifications include, but are not limited to, peptide nucleic acids (PNAs), phosphodiester group modifications (e.g., phosphorothioates, methylphosphonates), 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitutions of 4-thiouridine, 5-bromine or 5-iodo-uracil substitutions; Main chain modifications, methylation, unusual base pairing combinations, such as isobases, isocytidines and isoguanidines, etc. Nucleic acids may also include non-natural bases, such as nitroindole. Modifications may also include 3' modifications and 5' modifications, including but not limited to end-capping with fluorophores (e.g., quantum dots) or another portion.

[0045] "Polymerase" refers to an enzyme that performs template-directed synthesis of a polynucleotide (e.g., DNA and / or RNA). The term encompasses both full-length polypeptides and domains having polymerase activity. DNA polymerases are well known to those skilled in the art and include, but are not limited to, DNA polymerases isolated or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, or modified versions thereof. Additional examples of commercially available polymerases include, but are not limited to, Klenow fragment (New England Biolabs, Inc., Ill.), and PCR fragments. New England Inc.), Taq DNA polymerase (QIAGEN), 9°N TM DNA polymerase (New England Co., Ltd.), Deep Vent TM DNA polymerase (New England Co., Ltd.), Manta DNA polymerase Bst DNA polymerase (New England Inc.) and phi29 DNA polymerase (New England Biolabs, Inc.) Company Limited).

[0046] Polymerases include both DNA-dependent polymerases and RNA-dependent polymerases, such as reverse transcriptases. At least five families of DNA-dependent DNA polymerases are known, but most belong to families A, B, and C. Other types of DNA polymerases include bacteriophage polymerases. Similarly, RNA polymerases typically include eukaryotic RNA polymerases I, II, and III and bacterial RNA polymerases as well as bacteriophage and viral polymerases. RNA polymerases can be DNA-dependent and RNA-dependent.

[0047] As used herein, the term "partition" or "partitioned" refers to separating a sample into multiple parts or "partitions". Partitions are generally physical so that a sample in one partition will not or substantially will not mix with a sample in an adjacent partition. Partitions can be solid or fluid. In some embodiments, the partition is a solid partition, for example, a microchannel or micropore, or as described herein, a bead. In some embodiments, the partition is a fluid partition, for example, a droplet. In some embodiments, a fluid partition (e.g., a droplet) is a mixture of immiscible fluids (e.g., water and oil). In some embodiments, a fluid partition (e.g., a droplet) is an aqueous droplet surrounded by an immiscible carrier fluid (e.g., oil). Exemplary arrays of holes and hole descriptions can be found in, for example, U.S. Patents Nos. 9,103,754 and 10,391,493. Hole arrays (nanopores, micropores, hole groups) can be used to capture solid phase carriers, optionally in addressable known locations. Therefore, hole arrays can be configured to facilitate capturing beads in at least one or optionally a small group of solid phase carriers in a single solid phase carrier format. Exemplary microwell arrays and methods of delivering beads to microwells and analysis thereof are described, for example, in PCT / US2021 / 034152.

[0048] As used herein, a "barcode" is a short nucleotide sequence (e.g., at least about 4, 6, 8, 10, 12, 15, 20, 50 or 75 or 100 nucleotides long or longer) that identifies a molecule of a partition or sample to which it is conjugated or from which it is derived. For example, a barcode can be used to identify a molecule derived from a partition, or a bead that is sequenced later by a bulk reaction. For example, such a barcode may be unique to the partition or bead compared to barcodes present in other partitions or beads. For example, a partition containing target RNA from a single cell (which may be a hollow nanovial) may be subjected to reverse transcription conditions using primers containing different partition-specific barcode sequences in each partition, thereby incorporating a unique "cell barcode" (because different cells are in different partitions, and each partition has a unique partition-specific barcode) into the reverse transcribed nucleic acid of each partition. Therefore, nucleic acids from each cell can be distinguished from nucleic acids of other cells due to a unique "cell barcode". In some embodiments described herein, other barcodes uniquely identify the molecule to which they are conjugated, i.e., the barcode acts as a unique molecular identifier (UMI), or still other barcodes can indicate the sample of origin ("sample barcode"). The length of the underlying barcode sequence determines how many unique samples can be distinguished. For example, depending on degeneracy, 1 nucleotide barcode can distinguish 4 or fewer different partitions; 4 nucleotide barcodes can distinguish 4 4or 256 partitions or fewer; a 6-nucleotide barcode can distinguish 4096 different partitions or fewer; and an 8-nucleotide barcode can index 65,536 different partitions or fewer.

[0049] An "oligonucleotide" is a polynucleotide. Generally speaking, in some embodiments, an oligonucleotide will have less than 250 nucleotides, between 4-200, such as 10-150 nucleotides.

[0050] A "clonal" copy of a polynucleotide means that the copies are identical in sequence. In some embodiments, there are at least 100, 1000, 10 4 One or more clonal copies of an oligonucleotide attached to a bead.

[0051] The "3' capture sequence" on an oligonucleotide refers to the most 3' portion of the oligonucleotide. The capture sequence can be as little as 1-2 nucleotides in length, but more typically 6-12 nucleotides in length, and in some embodiments 4-20 or more nucleotides in length. The capture sequence can be fully complementary to the target nucleic acid (e.g., the 3' end of the target nucleic acid), but as will be understood in some embodiments and under certain conditions, 1, 2, 3, 4 or more nucleotides can be mismatched while still allowing the 3' capture sequence of the oligonucleotide to bond to the target nucleic acid. In other embodiments, conditions can be selected so that only fully complementary sequences will bond. The 3' capture sequence can be a random sequence, a poly T or poly A sequence, a target-specific sequence, or a universal sequence. For example, in embodiments where a transposon (e.g., a modified Tn5, such as a marker enzyme) inserts an end sequence to sample nucleic acid, the capture 3' end sequence can be complementary or identical to the added end.

[0052] Antibodies can exist as intact immunoglobulins or in any of a number of well-characterized fragments that include specific antigen binding activity. Such fragments can be produced by digestion with various peptidases. Pepsin digests the antibody below the disulfide bonds in the hinge region to produce F(ab)′2, i.e., Fab itself is bound to V by disulfide bonds. H -C H1 connected to the light chain dimer. F(ab)'2 can be reduced under mild conditions to destroy the disulfide bond in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region (see "Fundamental Immunology" (Paul ed., 3rd edition, 1993)). Although various antibody fragments are defined with respect to the digestion of intact antibodies, those skilled in the art will appreciate that such fragments can be synthesized de novo chemically or by using recombinant DNA methods. Therefore, as used herein, the term antibody also includes antibody fragments produced by modifying intact antibodies, or antibody fragments synthesized de novo using recombinant DNA methods (e.g., single-chain Fv) or antibody fragments identified using phage display libraries (see, e.g., McCafferty et al., "Nature" 348: 552-554 (1990)).

[0053] For the preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler and Milstein, Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96. Alan R. Liss, Inc. 1985). The technique for producing single-chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies against the polypeptides of the present invention. Furthermore, transgenic mice or other organisms such as other mammals can be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heterologous Fab fragments that specifically bind to a selected antigen (see, e.g., McCafferty et al., supra; Marks et al., Biotechnology 10:779-783 (1992)).

[0054] Antibodies bind to an "epitope" on an antigen. An epitope is a specific antibody binding interaction site on an antigen and may include several amino acids or portions of several amino acids, such as 5 or 6 or more amino acids, such as 20 or more amino acids, or portions of those amino acids. In some cases, an epitope includes, for example, non-protein components from carbohydrates, nucleic acids, or lipids. In some cases, an epitope is a three-dimensional portion. Thus, for example, where the target is a protein, an epitope may include continuous amino acids or amino acids approached by protein folding (e.g., discontinuous epitopes) from different parts of the protein. This is also true for other types of target molecules that form a three-dimensional structure.

[0055] The term "specific binding" refers to a molecule (e.g., an antibody or antibody fragment) that binds to a target with an affinity that is at least 2 times greater, e.g., at least 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 25 times, 50 times, or 100 times greater than the affinity of the molecule that binds to a non-target compound. For example, a specifically binding antibody αταργετ πρoτειv will typically bind to ταργετ πρoτsv with an affinity that is at least 2 times greater than the affinity of the antibody that binds to a non-target (e.g., other cellular proteins).

[0056] A "label" or "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., commonly used in ELISA), biotin, digoxigenin or haptens, as well as proteins or other entities that can be made detectable, for example, by incorporating a radioactive label into a peptide or antibody that is specifically reactive with the target peptide. Any method known in the art for conjugating antibodies to labels can be used, for example, using the methods described in Hermanson, Bioconjugate Technology ( Bioconjugate Techniques )》1996, Academic Press, Inc., San Diego. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG. 1 depicts barcoding with a barcode bead plug: A) Allow core-empty gel beads or nanovials to settle onto a 2D surface. B) Add cells to a solution and enter a nanovial by sedimentation and / or centrifugation. The cells attach to the base of the nanovial, C) Add barcode gel beads to a solution and the barcode gel beads enter the nanovial. The barcode gel beads also bind to the interior of the nanovial. D) Add cell lysis and reverse transcription reagents to the plug-cell-nanovial complex. The molecular weight of the reverse transcriptase is low enough to support diffusion throughout the hydrogel complex containing the nanovial and the barcode gel beads. The molecular weight of the RNA / DNA released from the lysed cells is high enough to be trapped within the complex. E) Use the barcode oligonucleotides on the barcode beads to initiate reverse transcription of RNA and production of barcoded cDNA. Prepare samples for NGS and sequence.

[0058] FIG. 2 depicts barcoding with a swollen barcode bead plug: A) Allow core-empty gel beads or nanovials to settle onto a 2D surface. B) Add cells to the solution and enter the nanovial by sedimentation and / or centrifugation. The cells attach to the base of the nanovial, C) Add barcode gel beads to the solution and the barcode gel beads enter the nanovial. The barcode gel beads also bind to the interior of the nanovial. D) Add gel bead swelling solution. The gel beads swell to create a plug at the orifice of the nanovial. Apply cell lysis and reverse transcription reagents. The molecular weight of the reverse transcriptase is low enough to support diffusion throughout the complex. The molecular weight of the RNA / DNA released from the lysed cells is high enough to be trapped within the complex. E) Use the barcode oligonucleotides on the barcode beads to initiate reverse transcription of RNA and production of barcoded cDNA. Prepare samples for NGS and sequence.

[0059] FIG. 3 depicts barcoding with multiple swollen barcode bead plugs: A) Allow core-empty gel beads or nanovials to settle onto a 2D surface. Cells are added to the solution and enter the nanovial by sedimentation and / or centrifugation. The cells attach to the base of the nanovial, C) A high concentration of barcode gel beads is added to the solution and the barcode gel beads enter the nanovial. The barcode gel beads also bind to the interior of the nanovial. D) Cell lysis and reverse transcription reagents are added to the plug-cell-nanovial complex. The molecular weight of the reverse transcriptase is low enough to support diffusion throughout the complex. The molecular weight of the RNA / DNA released from the lysed cells is high enough to be trapped within the complex. E) The barcode oligonucleotides on the barcode beads are used to initiate reverse transcription of RNA and barcoded cDNA production. Samples are prepared for NGS and sequenced.

[0060] FIG4 depicts barcoding with barcode beads and oil: A) Cells and beads are introduced into arrayed nanovials by gravity sedimentation or centrifugation. Although more than one gel bead per nanovial is depicted, the geometry of the nanovials and gel beads allows only a single gel bead to enter the nanovial. B) Oil is added to the mixture, and the cell-containing nanovial and bead solution is agitated to form droplets around the nanovial as shown in (C). D) The mixture is placed in a thermal cycler for barcoding, barcoded nucleic acid samples are prepared, and then NGS is performed.

[0061] Figure 5 depicts a multi-step process using a swollen barcode bead plug: A) Allow the core-empty gel beads or nanovial to settle onto a 2D surface. B) Add cells to the solution and enter the nanovial by sedimentation and / or centrifugation. The cells attach to the base of the nanovial, C) Add barcode gel beads to the solution and the barcode gel beads enter the nanovial. The barcode gel beads also bind to the interior of the nanovial. D) Add a gel bead swelling solution. The gel beads swell to create a plug at the orifice of the nanovial. E) Add a protein degradation reagent to denature and destroy all proteins, including histones that bind to DNA. During this step, high molecular weight nucleic acids cannot diffuse outside the core, where the cells were. After protein destruction is complete, the protein degradation solution is inactivated and / or washed away. F) Add a barcode encoding enzyme, followed by barcoding, sample preparation, and NGS.

[0062] Figure 6 Depicted are the layers of a possible nanovial having a PEG phase layer, a gelatin layer, and an oligonucleotide attached to the gelatin layer from bottom to top as depicted. The right side of the figure depicts an example in which a three-arm PEG spacer is attached to the gelatin.

[0063] Figure 7 Depicted is the use of click chemistry to attach cell barcode encoding oligonucleotides to nanovials. As shown, the NHS-PEG-azide three-arm linker (eg Figure 6 Linking of the oligonucleotides (shown as linked to gelatin) to DBCO-modified oligonucleotides can occur via a click chemistry reaction. DETAILED DESCRIPTION

[0064] introduce

[0065] It has been determined that the opening in the hollow nanovial containing the introduced cells can then be blocked by introducing hydrogel beads into the opening, thereby functionally making the nanovial a partition that retains large biomolecules such as RNA and DNA but reagents can diffuse therein to perform many molecular biological processes. For example, the cells in the blocked nanovial can be lysed, and the nucleic acid from the lysed cells can be connected to the barcoded oligonucleotides, which can be present in the nanovial or can be introduced (for example, connected thereto) by hydrogel beads. Therefore, for example, the single-cell nucleotide sequencing method that has been carried out in the droplet in the past can be carried out in the blocked nanovial as described herein instead. Examples of techniques that can be carried out in the blocked nanovial rather than other types of partitions include ATAC-seq and RNA-seq, etc. Similar methods can be applied to micropores, such as blocking the micropore openings with hydrogel beads, as explained herein about the nanovial openings. Similarly, the method using the blocked nanovial can be equally applied to blocked micropores.

[0066] Cells can be introduced into hollow nano vials or microwells as needed. For example, in some embodiments, cells are mixed with hollow nano vials. In some embodiments, the hollow nano vials are crescent-shaped (e.g., "C") and are allowed to settle on a planar surface. Due to the shape and eccentric weight of the hollow nano vials, their openings will face upward, and cells can be allowed to settle into the openings of the hollow nano vials. In some embodiments, the size of the opening and cavity of the nano vials is such that the average cavity can accommodate only one cell. In some embodiments, most (e.g., at least 50%, 60%, 70%, 80%, or 90% in the beads) contain only one cell, or optionally one or zero cells. This is achieved by loading 0.05-0.1 cells per nano vial on average. If there is a binding moiety (e.g., biotin or streptavidin) on the nano vial, the cell can be combined with the nano vial, as long as the corresponding binding partner is located on the outer membrane of the cell. Coating the substrate of the nanovial with one or more integrins or another cell binding agent is another way to provide a capture patch for binding to cells entering the nanovial cavity. In some embodiments, cell capture is irreversible. In some embodiments, the binding moiety is reversible, and thus cell capture is reversible.

[0067] Nanovials are typically hollow hydrophilic beads with openings for sample and reagent entry and exit. Exemplary hollow nanovials are described, for example, in de Rutte et al., bioRxiv Journal (bioRxiv) September 18, 2020 ( ht tp s： / / doi.org / 10.1101 / 2020.03.09.984245); de Rutte, ACS Nano (2022) https: / / doi.org / 10.1021 / acsnano.1c11420; and U.S. Patent Publication No. 2021 / 0268465 (describing the hollow beads as "droplets" or "drop-borne particles"). The diameter of the hollow beads can be, for example, between about 10 μm and about 500 μm, for example, between about 20 μm and 200 μm, for example, between 40 μm and 20 μm. In some embodiments, the hollow beads are crescent-shaped so that the hollow beads have a tendency to settle under gravity when their openings are facing upward. This is because the center of gravity is offset from the center in the direction opposite to the opening of the nanovial. The beads include a void volume or cavity therein. The void or cavity defines a three-dimensional volume that holds at least a portion of a dispersed phase solution or fluid (e.g., an aqueous phase). Exemplary fluid volumes contained within voids or cavities may be, for example, about 100 fL-10 nL. The length dimension of voids or cavities within beads (eg, diameter of spherical voids) may be several microns, for example greater than about 5 μm and less than about 250 μm.

[0068] In some embodiments, the hollow nanovial is produced by flowing two phases to form an emulsion to produce a hollow nanovial. Microgel beads containing cavities can be manufactured, for example, using an aqueous two-phase system (ATPS) combined with droplet microfluidics. See, for example, U.S. Patent Publication No. US 2021 / 0268465; S. Ma et al. Small. 8, 2356-2360 (2012); BD Fairbanks et al., Aversatile synthetic extracellular matrix mimic via thiol-norbornene photopolymerization. Advanced Materials (Adv. Mater.) (2009). In some embodiments, one phase of the aqueous two-phase system includes a crosslinkable component, while the other phase does not contain a crosslinkable component. In some embodiments, a cross-linkable PEG phase and a dextran phase are co-flowed in a microfluidic droplet generator together with a third oil phase containing a surfactant to produce a mixed aqueous emulsion in which a uniform fraction of the PEG phase and the dextran phase is present in each droplet suspended in the oil phase. In other embodiments, gelatin is used instead of dextran. For example, in some embodiments, a cross-linkable PEG phase and a gelatin phase are co-flowed in a microfluidic droplet generator together with a third oil phase containing a surfactant to produce a mixed aqueous emulsion in which a uniform fraction of the PEG phase and the gelatin phase is present in each droplet suspended in the oil phase. See, for example, Lee, American Chemical Society Nano 2022, 16, 38-49. In some embodiments, separate prepolymer solutions of poly(ethylene glycol) diacrylate (PEGDA) and poly(propylene glycol) diacrylate (PPGDA) are co-flowed, formed into the desired cross-sectional morphology in the microchannel flow in the flow, and then photo-cross-linked. See, e.g., Wu et al., Sci. Adv. 2020. The same reference shows that other continuous phases that are immiscible with water can also be used, including poly(dimethylsiloxane-co-diphenylsiloxane) (PSDS), PPG, decanol, and toluene.

[0069] Nano vials (or microwells), hydrogel beads, or both can be connected to oligonucleotides, the oligonucleotides comprising barcodes that identify nano vials and / or hydrogels, respectively, and the barcodes serve as partition-specific barcodes. In some embodiments, hollow nano vials are connected or otherwise associated with multiple cloned cell barcode encoding oligonucleotides having 3' capture sequences. In some embodiments, cell barcode encoding oligonucleotides can be connected to hydrogel beads as needed (see below). The method of connecting cell barcode encoding oligonucleotides to hydrogel beads or nano vials will depend on the composition of the beads. In some embodiments, the 5' end of the cell barcode encoding oligonucleotide is connected to the beads, or the cell barcode encoding oligonucleotide is otherwise connected so that the 3' capture sequence can be used for hybridization with a complementary sequence.

[0070] In some embodiments, the cell barcode encoding oligonucleotide is connected to the nanovial through a spacer, thereby allowing the barcode encoding oligonucleotide to be more available for bonding to complementary sequences. In some embodiments, the spacer allows more than one barcode encoding oligonucleotide to be connected to the spacer, thereby allowing a higher density of barcode encoding oligonucleotides on the nanovial. For example, in some embodiments, a 3-arm polyethylene glycol (PEG) with terminal NHS groups can be used for the gelatin cavity of the nanovial, and the PEG will double the functional groups on the cavity surface for oligonucleotide attachment. An exemplary three-arm PEG spacer is shown in Formula I

[0071]

[0072] In some embodiments, click chemistry can be used to attach the cell barcode encoding oligonucleotide to the nanovial or bead. For example, NHS-PEG-azide can be attached to the amine (NH2) moiety in the cavity or elsewhere on the nanovial, followed by the attachment of the DBCO modified oligonucleotide to the azide group via click chemistry. See Figure 7-8. Click chemistry involves an azide alkyne Huisgen cycloaddition reaction in which, in some embodiments described herein, a first oligonucleotide having a terminal alkyne moiety (e.g., but not limited to DBCO modified) is reacted with a spacer having a terminal azide moiety and promoting or catalyzing a reaction between a moiety to covalently attach the two oligonucleotides. In some embodiments, the azide-alkyne Huisgen cycloaddition is a 1,3-dipolar cycloaddition between an azide and a terminal or internal alkyne to obtain a 1,2,3-triazole. For example, in some embodiments, the reaction can be catalyzed by copper (copper (I) catalyzed azide-alkyne cycloaddition (CuAAC)), or promoted by strained difluorocyclooctyne (DIFO) (strain promoted azide-alkyne cycloaddition (SPAAC)). See, e.g., Chemical Reviews: Click Chemistry, June 23, 2021, Vol. 121, No. 12, pp. 6697-7248, including, e.g., Fantoni et al., "A Hitchhiker's Guide to Click-Chemistry with Nucleic Acids," Chem. Rev. 2021, 121, 7122-7154. In some embodiments, click ligation can be performed by incubating at 45° C. for, e.g., 2 hours in the presence of vitamin C, Cu(II)-TBTA, MgSO4, THPTA, and DMSO.

[0073] In other embodiments, the partition-specific barcode-encoded oligonucleotides are not connected or associated with the hollow nanovial, and in some of these embodiments, the oligonucleotides can be delivered to the cavity within the hollow nanovial via hydrogel beads as described in more detail below.

[0074] In some embodiments, sets of hollow nanovials can be connected or otherwise associated with the same hash oligonucleotide, wherein different sets have different hash oligonucleotides, thereby allowing different nanovial sets to be tracked by hash barcodes in the hash oligonucleotides. These sets will be used, for example, for cell hashing, for example, as described in, for example, Stoeckius et al., Genome Biology (2018) 19: 224. The hash oligonucleotide may include, for example, a 5′ PCR handle sequence that allows a universal primer to bind to a sequence, such as a hash barcode sequence that identifies a nanovial set relative to other nanovials; and (i) a 3′ sequence that is complementary to a 3′ sequence downstream of a barcode segment in a partition-specific barcode encoding oligonucleotide; and / or (ii) a splint sequence that allows the hash oligonucleotide to be later attached to a partition-specific barcode encoding oligonucleotide. In some embodiments, attachment may include hybridizing the hash oligonucleotide directly to a partition-specific barcode encoding oligonucleotide with or without primer-templated DNA synthesis. In some embodiments, attachment can include hybridizing the hash oligonucleotide indirectly to the partition-specific barcode encoding oligonucleotide by hybridization with the splint oligonucleotide with or without ligation. In some embodiments, attachment can include ligating the hash oligonucleotide to an amplicon formed by primer-templated hybridization extension and / or ligation of the partition-specific barcode encoding oligonucleotide and the target nucleic acid from the lysed cells. In some embodiments, the nanovial can be ligated to the hash oligonucleotide and the partition-specific barcode encoding oligonucleotide.

[0075] Hash oligonucleotides can be used to provide the marking of nano vial sets. The nano vial sets can receive different samples, for example, so as to allow hash oligonucleotides to mark different samples, which can be mixed and tracked by bar code encoding afterwards. In some embodiments, the nano vial sets can receive different aliquots of cells from the same sample, for example, so as to allow hash oligonucleotides to mark different aliquots from the same sample, which can be mixed and tracked by bar code encoding afterwards. In some embodiments, the nano vial sets can receive different aliquots of cells from different samples and from the same sample, which can be mixed and tracked by bar code encoding afterwards. In some embodiments, as described herein, cells can be introduced into nano vials comprising hash oligonucleotides, and different sets with different hash oligonucleotides can be combined and blocked (the order can be blocking and combination or combination and blocking). Cell nucleic acid can then be subjected to partition-specific bar code encoding, and hash oligonucleotides can be connected to the generated partition-specific bar code-encoded sample nucleic acid. The nucleotide sequences can then be used to track sequences from different sets of nanovials by hashing oligonucleotides, and specific partitions can be tracked by partition-specific barcodes.

[0076] Any type of cell can be introduced into the hollow nano vial (or microwell). In some embodiments, the cell is a eukaryotic cell, such as a human, mouse, rat or other mammalian cell. In some embodiments, the cell is an immune cell, such as, but not limited to, a hybridoma, a T cell or a B cell. In some embodiments, the cell is a bacterial or fungal or plant cell.

[0077] After the cells are introduced into the hollow nano vial (or micropore), a blockage can be formed in the opening in the bed, thereby preventing the cells from spreading from the nano vial (or micropore). For example, in some embodiments, although the cells and the cell products from the lysed cells can easily spread from the nano vial opening, after the opening is blocked, the cells and the cell products will not basically escape from the nano vial, thereby allowing the use of the blocked nano vial as a partition, without the need for droplets, micropores or other partitioning techniques. This can be achieved, for example, by introducing one or more hydrogel beads into the opening in the hollow nano vial, thereby preventing the cells from leaving the cavity in the hollow nano vial. In some embodiments, the hydrogel beads are introduced into the opening enough to block the opening, and the intact cells in the nano vial will not be released, for example. The hydrogel beads can be introduced into the nano vial as needed, for example, the beads can be allowed to settle in the nano vial by gravity or by centrifugation. In other embodiments, after the hydrogel beads are introduced into the opening in the nanovial, one or more agents or condition changes (e.g., temperature changes) are introduced to swell the hydrogel beads, thereby reducing the ability of the hydrogel beads to escape from the opening and fill the gap between the interior of the nanovial and the surface of the gel beads. In some embodiments, the hydrogel beads comprise polyacrylamide, and swelling of the beads can be induced by contacting the beads with ethanol.

[0078] In some embodiments, the hydrogel beads may include a component that causes swelling when cut, thereby blocking the opening of the nano vial. For example, in some embodiments, the beads include a component that cleaves when contacted with a reducing agent. An example of this component is, for example, polyacrylamide cross-linked with bisacrylcystamine. Exemplary reducing agents can be applied to cleave disulfide bonds in the joint. Exemplary reducing agents can include, but are not limited to, dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine (TCEP) or other reducing agents.

[0079] In other embodiments, the hydrogel comprises a photocleavable component. Upon exposure to light or light of a wavelength of the photocleavable component, the beads will swell such that when present in the opening of the nanovial, the nanovial opening will be blocked. In some embodiments, the photocleavable element may be incorporated into the hydrogel bead matrix, for example, through one or more acrylate groups. In some embodiments, the photocleavable component is an acrylate functionalized dextran. In some embodiments, the beads comprise acrylamide or other hydrogel components that comprise a photocleavable crosslinker, such as o-nitrobenzyl (o-NB) diacrylate.

[0080] Additionally or alternatively, a low salt buffer or pure water can be used to swell the beads, while a high salt buffer can be used to shrink the beads, which can be induced, for example, to allow the beads to enter the nano vial. An exemplary high salt buffer that can shrink the hydrogel gel beads is, for example, 100 mM MgCl2. An exemplary low salt buffer that can swell the beads is, for example, 10 mM Tris 1 mM EDTA. In another embodiment, a polyethylene glycol (PEG) solution (e.g., 15% w / v PEG 6000) will also shrink the beads. In this case, if the PEG solution is replaced by a solution that does not contain PEG or contains a lower concentration of PEG, the beads will swell subsequently.

[0081] In certain embodiments, blocking prevents cell products and the cell itself from escaping. For example, macromolecules such as polynucleotides and proteins will not be able to diffuse significantly from the cavity in the blocked nano vial. Optionally, if it is desired that specific cell products and / or nucleic acid species escape, those substrates can be initially cut to produce a relatively low molecular weight product that diffuses through the nano vial-gel bead complex. The successful blocking of the nano vial can be measured in any manner to detect the ability of the nano vial to retain molecules compared to an open nano vial (for example, not receiving hydrogel beads). In certain embodiments, species mixing experiments can be performed to measure blocking. For example, mouse and human cells can be loaded in nano vials. In bar coding, sample preparation and next generation sequencing, the amount of nucleic acid from species not contained in the nano vial can be a measure of the degree to which the plug-nano vial complex prevents nucleic acid from moving from one nano vial to another nano vial. This is referred to as a "cell purity" metric.

[0082] Hydrogel beads of various compositions have been described previously. It will be appreciated that hydrogels are hydrophilic, and thus in some embodiments, "nano-vials" and "hydrogel beads" may be composed of the same material, while in other embodiments, the two are composed of different materials. In any case, to maintain clarity between hollow beads containing cells and beads that block openings, the terms "hollow nano-vials" or "nano-vials" are used to describe the former, while "hydrogel beads" are used to describe the latter.

[0083] Hydrogel beads can be made of various materials. Exemplary hydrogel beads can be, for example, agarose hydrogel beads or polyacrylamide hydrogel beads. In other embodiments, the hydrogel comprises, for example, polystyrene, silica, polymethyl methacrylate, alginate, polyethylene glycol (PEG), nylon and / or other polymers that are cross-linked or not. Other hydrogels include, but are not limited to, for example, U.S. Patent No. 4,438,258; No. 6,534,083; No. 8,008,476; No. 8,329,763; U.S. Patent Application No. 2002 / 0,009,591; No. 2013 / 0,022,569; No. 2013 / 0,034,592; and International Patent Publication No. WO / 1997 / 030092; and the hydrogel described in No. WO / 2001 / 049240. In general, the size of the hydrogel beads can be selected as described herein to allow the hydrogel beads to enter the opening in the hollow nanovial, but large enough to block the opening. The hydrogel beads can also be sensitive to shrinking agents and swelling agents as described herein. The matrix percentage of the hydrogel determines the porosity of the hydrogel and can be selected to prevent target molecules from moving outside the nanovial gel bead complex while allowing buffer and low molecular weight proteins to diffuse freely. Hydrogels greater than 3% hydrogel material generally capture high molecular weight nucleic acids, but allow low molecular weight enzymes, ions and detergents to diffuse.

[0084] In some embodiments, a population of hydrogel (e.g., acrylamide cross-linked with bisacrylamide) functionalized beads (e.g., containing functionalized oligonucleotides attached to the hydrogel beads) sized to be slightly (e.g., 0.5%-25%) larger than the opening in the hollow nanovial can be exposed to a buffer (e.g., 10 mM MgCl2) containing a material that produces functionalized beads of a smaller equilibrium size, thereby causing the majority to shrink to a size smaller than the size of the hollow nanovial opening, thereby loading the functionalized beads into the hollow nanovial opening, and then changing the surrounding buffer (e.g., to a lower salt buffer, such as Tris EDTA buffer, etc.) to produce swelling, but wherein the functionalized beads remain in the hollow nanovial opening. This process effectively closes the hollow nanovial opening by one or more semi-permeable functionalized beads.

[0085] In some embodiments, in addition to blocking the opening in the nano vial (or micropore), the hydrogel beads can also deliver oligonucleotides to the cavity of the nano vial (or micropore), for example, to attach to the target cell nucleic acid in the cavity. Oligonucleotides can be connected to beads as needed. The solid support surface of the beads can be modified to include a joint for attachment to the barcode oligonucleotide. The joint can include a cleavable portion. Non-limiting examples of cleavable portions include disulfide bonds, dioxyuridine moieties, and restriction enzyme recognition sites. The method of connecting oligonucleotides to beads is described in, for example, WO 2015 / 200541. In some embodiments, the oligonucleotides configured to connect hydrogel beads to barcodes are covalently linked to the hydrogel. Many methods for covalently linking oligonucleotides to one or more hydrogel matrices are known in the art. As only one example, aldehyde-derivatized agarose can be covalently linked to the 5′-amine group of synthetic oligonucleotides.

[0086] Optionally, multiple nano vials (many but not all of which contain cells) can be enriched for beads containing cells. This can be achieved, for example, when cells emit fluorescent signals, thereby allowing beads to be sorted by FACS. Alternatively, methods such as differential sedimentation or centrifugation can be used to enrich beads containing cells at a higher density. Fluorescent signals can be accumulated throughout the nano vial, throughout the gel beads, and / or in a specific location throughout the nano vial gel bead complex (such as at the base of the nano vial opening).

[0087] After the opening in the hollow nano vial (or micropore) is blocked, the cells in the nano vial (or micropore) can be lysed. Lysis can be induced by temperature or the introduction of one or more reagents that cause cell lysis. Exemplary lysing agents can include, for example, detergents (e.g., SDS), protein denaturants, and / or proteases. Due to the properties of the nano vial and the obstructive hydrogel beads, small molecules can diffuse into the cavity of the nano vial. Therefore, the lysing agent can be incubated with the nano vial, thereby allowing the lysing agent to diffuse into the cavity of the bed where the cell resides. In certain embodiments, the protein denaturant includes guanidine. Protein denaturants and / or proteases can be used to remove histones from chromosomal DNA, for example, when the cell DNA is the target cell nucleic acid. Optionally, after lysis, one or more washing / rinsing steps can be performed on the nano vial to allow the lysing agent to diffuse after the lysis occurs, thereby leaving larger molecules from the lysed cells in the cavity of the nano vial.

[0088] Macromolecules such as DNA and RNA from lysed cells (e.g., nucleic acid species greater than 1 kbp (650 KDa)) will remain substantially in the cavity of the nanovial, allowing molecular methods to be applied to the nucleic acids, thereby substantially separating them from the nucleic acids in the individual nanovials. On the other hand, smaller molecules including, for example, polymerases, can be diffused into the cavity of the nanovial, allowing various molecular reactions to be triggered. Thus, for example, an oligonucleotide present in the cavity of the nanovial (e.g., introduced, connected to the nanovial, or delivered via hydrogel beads) can be connected to one or more nucleic acids from the cell. The nucleic acid can be, for example, RNA or DNA.

[0089] Oligonucleotides can be used to perform primer extension or other hybridization-based reactions. In some embodiments, the 3' end sequence of the oligonucleotide is directly bonded to the target nucleic acid. For example, if the target nucleic acid is mRNA, the 3' end sequence can be a polydT sequence (e.g., 6-20 continuous dT nucleotides), or the 3' end can include a random body (e.g., a random sequence of 6-10 or more nucleotides) for randomly priming the target, or the 3' end sequence can be gene-specific so that one or more target nucleic acids are specifically amplified. In some embodiments, the 3' sequence of the oligonucleotide is bonded to a universal sequence on the target nucleic acid. For example, enzymatic fragmentation can cause an adapter sequence to be inserted into the end of the fragmented nucleic acid, and the 3' end can be reverse complementary to the adapter sequence. In some embodiments, the oligonucleotide can be connected to the cell DNA or used as a primer in a primer extension reaction using a DNA polymerase and the cell DNA as a template to form a DNA molecule comprising at least a portion of the oligonucleotide sequence and the cell DNA. In any of these embodiments, the oligonucleotide may be attached at its 5' end to a solid support (eg, any bead), or may be released from the support into solution in a cavity prior to attachment to cellular nucleic acid.

[0090] In any of the embodiments described herein, the oligonucleotides connected to the beads may include multiple cloned copies of one or more barcode nucleotide sequences. In some embodiments, the oligonucleotides include a barcode sequence that is unique to the beads to which they are attached, and can thereby be used to distinguish the oligonucleotides from different beads, for example, after the oligonucleotides are released and used to generate sequencing reads. For example, most or substantially all cellular nucleic acids connected to the oligonucleotides will include the same barcode sequence, or in the case where more than one hydrogel bead has introduced more than one oligonucleotide barcode (one barcode clone set per bead in the cavity), there may be two or more different barcode sequences connected to different nucleic acids from the same cell, thereby requiring downstream deconvolution to remove or decrypt nucleic acids encoded by different barcodes from the same cell. Additional barcodes (such as, but not limited to, unique molecular identifiers (UMIs)) or sample-specific barcodes may also be included in the oligonucleotide sequence, and thus connected to cellular nucleic acids.

[0091] Optionally, the blocked nanovial can be introduced into a droplet or other partition for further manipulation. In other embodiments, the blocked nanovial is not introduced into a droplet or other partition, and the blocked nanovial itself is employed as a partition.

[0092] After completing the molecular reaction as described above in the hollow nanovial (or microwell) (e.g., the barcode has been attached to the cell nucleic acid), the contents of the nanovial (or microwell) can be combined into a bulk solution. In some embodiments, the hydrogel beads blocking the opening of the nanovial (or microwell) or the nanovial itself or both are digested or otherwise degraded to allow the contents of the nanovial (or microwell) to be released. This can be achieved, for example, by applying heat or one or more reagents that degrade or dissolve the hydrogel beads or the nanovial or both, thereby releasing the contents. In some embodiments, the hydrogel beads contain polyacrylamide and N,N'-bis(acryloyl)cystamine as a reversible crosslinker, which dissolves when reacting with a reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) or other reducing agents. In some embodiments, the solvent surrounding the nanovial is changed to shrink the hydrogel beads and thereby release the contents of the nanovial. As mentioned above, the hydrogel beads can be induced to shrink in the presence of, for example, ethanol, PEG (eg, 15%), and / or 100 mM MgCl2.

[0093] The nucleic acid can then be nucleotide sequenced. After the nucleic acid has been tagged with an oligonucleotide, the tagged nucleic acid can be prepared for nucleotide sequencing as needed. For example, universal priming sequences can be added to both ends of the tagged sequence (one universal priming sequence on each end). Any nucleotide sequencing method can be used as needed, as long as at least some of the DNA segment sequence and the barcode sequence are determined. Methods for high-throughput sequencing and genotyping are known in the art. For example, such sequencing technologies include, but are not limited to, pyrophosphate sequencing, ligation sequencing, single molecule sequencing, synthetic sequence (SBS), massively parallel cloning, massively parallel single molecule SBS, massively parallel single molecule real-time, massively parallel single molecule real-time nanopore technology, etc. Morozova and Marra provide a review of some such technologies in Genomics, 92: 255 (2008), which is incorporated herein by reference in its entirety.

[0094] Exemplary DNA sequencing techniques include fluorescence-based sequencing methods (see, e.g., Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor Laboratory, New York; the document is incorporated herein by reference in its entirety). In some embodiments, automated sequencing techniques as understood in the art are utilized. In some embodiments, the present technology provides parallel sequencing of partitioned amplicons (PCT Publication No. WO 2006 / 084132, the document is incorporated herein by reference in its entirety). In some embodiments, DNA sequencing is achieved by parallel oligonucleotide extension (see, e.g., U.S. Pat. Nos. 5,750,341; and 6,306,597, both of which are incorporated herein by reference in their entirety). Additional examples of sequencing techniques include Church polony techniques (Mitra et al., 2003, Analytical Biochemistry). Biochemistry, 320, 55-65; Shendure et al., 2005 Science, 309, 1728-1732; and U.S. Pat. Nos. 6,432,360; 6,485,944; and 6,511,803; the entireties of which are incorporated herein by reference); 454 pico-titer pyrosequencing (Margulies et al., 2005 Nature, 437, 376-380; U.S. Publication No. 2005 / 0130173; the entireties of which are incorporated herein by reference); Solexa single base addition technology (B Brennett et al., 2005, Pharmacogenomics, 6, 373-382; U.S. Pat. Nos. 6,787,308; and 6,833,246; the entireties of which are incorporated herein by reference); Lynx massively parallel tag sequencing technology (Brenner et al. (2000). Nat. Biotechnol. 18: 630-634; U.S. Pat. Nos. 5,695,934; and 5,714,330; the entireties of which are incorporated herein by reference); and Adessi PCR colony technology (Adessi et al. (2000). Nucleic Acids Res. 28, E87; WO 2000 / 018957; the entireties of which are incorporated herein by reference).

[0095] In general, high-throughput sequencing methods share the common features of massively parallel high-throughput strategies with the goal of reducing costs compared to older sequencing methods (see, e.g., Voelkerding et al., Clinical Chem., 55:641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7:287-296; each of which is incorporated herein by reference in its entirety). Such methods can be broadly divided into methods that typically use template amplification and methods that do not use template amplification. Methods that require amplification include pyrosequencing commercialized by Roche with 454 technology platforms (e.g., GS20 and GS FLX), Solexa platforms commercialized by Illumina, and the Supported Oligonucleotide Ligation and Detection (SOLiD) platform commercialized by Applied Biosystems. Non-amplification methods (also known as single-molecule sequencing) are exemplified by the HeliScope platform commercialized by Helicos BioSciences and platforms commercialized by VisiGen, Oxford Nanopore Technologies Ltd., Life Technologies / IonTorrent, and Pacific Biosciences, respectively.

[0096] The practice of the present invention can employ conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology, which are within the skill of the art. Such techniques are fully explained in the literature. See, e.g., Gennaro, AR, ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Hardman, JG, Limbird, LE and Gilman, AG, eds. (2001) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill Co.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press, Inc.; Weir, DM and Blackwell, CC, eds. (1986) Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publishers. Publications); Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel, FM et al., eds. (1999-2010) Current Protocols in Molecular Biology, John Wiley & Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; Newton, CR and Graham, A., eds. (1997) PCR (Introduction to Biotechniques Series), 2nd ed., Springer Verlag; Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.) (1989).

[0097] In some embodiments, after the sequencing reads are generated, the sequencing reads can be deconvoluted to merge sequencing reads with different barcodes but originating from the same partition. Therefore, in some embodiments, the method described above can be used, which involves determining the percentage of shared identical fragments and combined sample reads to assume that all such reads are from the same partition when the percentage of identical fragments exceeds a threshold, such as described in U.S. Patent Publication 2020 / 0056231. Other methods for deconvoluting sequencing reads (determining whether the reads are from the same partition or different partitions) may include, for example, the method described in PCTWO2017 / 120531. For example, in some embodiments, a method may involve providing a substrate comprising a barcode sequence or a repeated clone barcode sequence in a partition; and in the partition, a first particle conjugated to an oligonucleotide primer comprising a first barcode sequence and a second particle conjugated to an oligonucleotide primer comprising a second barcode sequence are associated with the barcode sequence from the substrate; thereby generating a nucleic acid signature of the particles in the partition, which can be used to distinguish a separate barcode from a barcode in the same partition (see PCT WO2017 / 120531). In other aspects for deconvoluting sequencing reads (determining whether the reads are from the same partition or different partitions), the method may include: forming partitions of the target nucleic acid, the partitions comprising a forward primer or its reverse complement, the forward primer comprising a barcode and a capture sequence complementary to the 3′ sequence, wherein different partitions contain different forward primers and partition ID tag oligonucleotides, the different forward primers comprising different barcode sequences, the partition ID tag oligonucleotides comprising the reverse complement of the capture sequence and a variable partition ID tag sequence; in the partitions, hybridizing at least one forward primer to the partition ID tag oligonucleotide to form a hybridized product; amplifying the hybridized products to form amplicons, wherein at least some of the amplicons are formed by the forward primer and the partition ID tag oligonucleotide; and sequencing the amplicons, wherein different forward primers are considered to be from the same partition if they form amplicons with the same variable partition ID tag sequence. In some of these embodiments, the forward primer and the partition ID tag oligonucleotide are connected to the same substrate when delivered to the partition; or the partition ID tag oligonucleotide has a closed 3' end so that the polymerase cannot extend the closed 3' end during amplification; or the partition ID tag oligonucleotide comprises a double-stranded variable partition ID tag sequence and one or two single-stranded 3' ends, wherein the one or two single-stranded 3' ends comprise the reverse complement of the capture sequence. See, e.g., PCT / US2019 / 015638.

[0098] The blocked nano bottle or micropore can also be used to detect proteins expressed by cells and optionally secreted from cells. For example, in some embodiments, one or more binding agents specifically bound to a cell target protein (for example, having affinity thereto) can be connected to a nano bottle or micropore. In certain embodiments, different target proteins have different (for example, 2 kinds, 3 kinds, 4 kinds or more kinds) binding agents. Cells can be subsequently loaded into nano bottle or micropore as described above, and openings can be blocked with one or more hydrogel beads, as described above. Proteins secreted from cells can be accumulated in nano bottle or micropore, and will be combined with affinity agent, thus substantially preventing target protein from diffusing from nano bottle or micropore. In certain embodiments, cell lysis can be induced as described above, thereby allowing target protein to be released from cells, thereby allowing target protein to be combined with affinity agent.

[0099] Then the target protein bound by the affinity agent in the blocked nano vial or micropore can be specifically detected. In some embodiments, for example, the second binding agent of direct or indirect labeling is introduced into the blocked nano vial or micropore by diffusion, and the presence, absence or quantity of the target protein can be detected. This can produce a sandwich assay format for detecting the target protein. In some embodiments, the signal from the second binding agent of the combination is sufficient to allow the nano vial to be sorted (for example, FACS sorting), thereby allowing the target protein to be detected. In some embodiments, with or without sorting, the excess unbound second binding agent from the nano vial or micropore can be washed, followed by specific detection of the signal from the label on the second binding agent. As described herein, in some embodiments, the first binding agent, the second binding agent or two binding agents are antibodies or other binding molecules, such as streptavidin or biotin. In some embodiments, the label is fluorescent. In some embodiments, there are a variety of (for example, 2, 3, 4 or more) different second binding agents, each second binding agent has a separate distinguishable label, thereby allowing separate detection (and optionally sorting) of different target proteins.

[0100] Compositions as described herein or kits for performing the described methods are also provided. Exemplary compositions may include, for example, a plurality of hollow nanovials (or microwells) containing one or more intact or lysed cells and one or more hydrogel beads blocking the openings in the nanovials (or microwells).

[0101] Prophet Example

[0102] Nano vials with a diameter of 35 microns across the opening and also having an integrin patch at the base of the internal cavity were suspended in PBS, 0.1% BSA, 0.1% F68 pluronic, and 15% PEG 6000. The nano vials were allowed to settle by gravity in a 2D plane located at the bottom of a petri dish. Since the center of gravity is positioned away from the opening, the nano vials settle with the opening facing upward. B cells also suspended in PBS, 0.1% BSA, 0.1% F68 pluronic, and 15% PEG 6000 were allowed to settle by gravity into the nano vials. After the cells fell to the base of the opening, the cells bound to the nano vials by attaching to the integrin. Cells were loaded at a lambda of <0.06 so that most of the nano vials had one cell or no cells. Thirty-two micron diameter clonal barcode gel beads suspended in PBS, 0.1% BSA, 0.1% F68 pluronic, and 15% PEG 6000 were also added to the petri dish and allowed to settle by gravity into the inner cavity of the nanovial. There was only enough space to load one gel bead per nanovial, so the gel beads did not have to be loaded at a lower lambda. The end result was that >80% of the nanovials would contain a single barcode gel bead. These clonal barcode gel beads were then swollen by exchanging the buffer with a solution that did not contain PEG and the opening was sealed. A lysis reagent containing 1% NP-40 was added to the buffer. The non-ionic detergent was flowed to the center of the nanovial cavity where the cells were located, thereby lysing the cells. High molecular weight DNA and RNA were trapped in the space between the nanovial and the gel beads because the pore size in the nanovial and the gel beads was too small to allow the molecules to diffuse. The nanovial-gel bead complex containing the captured RNA and DNA is washed to remove cell lysate, which is an inhibitor of downstream molecular biology reactions. A small molecular weight reverse transcriptase is then added to the solution along with a master mix buffer containing nucleotides. Due to the small molecular weight and thus small size of the enzyme, the enzyme and master mix diffuse into the captured DNA and RNA. After the poly A tail of the mRNA binds to the poly T primer that is part of the barcode oligonucleotide, reverse transcription occurs, thereby barcoding the RNA from a single cell. In order to release the barcoded RNA, PEG is then added back to the solution at 15%. The gel beads shrink due to the presence of PEG, allowing the barcoded cDNA to diffuse freely. Sample preparation is completed in batches, followed by sequencing. Single-cell RNA can be grouped, and thus the single-cell transcriptome can be reconstructed by binning the sequence reads according to the cloned barcode sequence.

[0103] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes thereto will occur to those skilled in the art and are included within the spirit and scope of the present application and the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A method for ligating an oligonucleotide to a cellular nucleic acid in a hollow nanovial, the method comprising: The cells are loaded into the opening in the hollow nanovial; inserting one or more hydrogel beads into the opening, thereby blocking the opening and preventing the cells from diffusing from the opening; lysing the cells in the hollow nanovial; One or more oligonucleotides are linked to the nucleic acids produced by the lysed cells, thereby barcoding the nucleic acids, wherein the oligonucleotides are present in the hollow nanovial prior to the loading or are introduced into the hollow nanovial via the hydrogel beads. 2 . The method of claim 1 , wherein the blocking comprises inducing swelling of the one or more hydrogel beads inserted into the nanovial.

3. The method of claim 1, wherein the blocking comprises inducing swelling of the nanovial.

4. The method of claim 1, wherein a majority of the nanovials contain one or zero cells.

5. The method of claim 1, wherein the oligonucleotide is attached to the hydrogel bead and, optionally, released from the hydrogel bead after sealing.

6. The method of claim 5, wherein the oligonucleotide is attached to the hydrogel bead and optionally released from the hydrogel bead prior to said attachment.

7. The method of claim 5, wherein the oligonucleotide has a barcode sequence, wherein individual hydrogel beads contain cloned copies of the oligonucleotide, and wherein the barcode sequence attached to an individual hydrogel bead is unique such that the barcode distinguishes the hydrogel bead from other hydrogel beads.

8. The method of claim 1, wherein the oligonucleotide has a barcode sequence, wherein an individual nanovial contains a cloned copy of the oligonucleotide, and wherein the sequenced barcode attached to an individual nanovial is unique such that the barcode distinguishes the nanovial from other nanovials.

9. The method of claim 1, further comprising extracting the barcoded nucleic acids from the nanovials, and combining the barcoded nucleic acids from different nanovials into a bulk solution, and performing nucleotide sequencing on the barcoded nucleic acids from the bulk solution.

10. The method of claim 1, wherein the lysing comprises diffusing one or more lysing agents into the cells in the nanovial.

11. The method of claim 10, wherein the lysis reagent is removed from the nanovial or inactivated prior to the ligating.

12. The method of claim 1, wherein the lysing comprises diffusing a protein denaturing and or degrading agent into the cells in the nanovial.

13. The method of claim 12, wherein the lysis reagent is removed from the nanovial or inactivated prior to the ligating.

14. The method of claim 1, wherein said ligating comprises ligating said oligonucleotide to said nucleic acid and optionally performing a template-dependent extension of said oligonucleotide using said nucleic acid as a template.

15. The method of claim 1, wherein the ligating comprises diffusing one or more ligating reagents into a sealed nanovial containing the lysed cells.

16. The method of claim 14, wherein the nucleic acid is DNA and the ligation reagent comprises a DNA polymerase or a ligase.

17. The method of claim 14, wherein the nucleic acid is RNA and the ligation reagent comprises a reverse transcriptase.

18. The method of claim 1, wherein the ligating comprises ligating the oligonucleotide to the nucleic acid by a click chemistry reaction.

19. The method of claim 1, wherein the hollow nanovial comprises dextran and polyethylene glycol (PEG).

20. The method of claim 1, wherein the hydrogel beads comprise polyacrylamide, polystyrene, silica, polymethylmethacrylate, alginate, polyethylene glycol (PEG), nylon, or agarose.

21. The method of claim 1, wherein the hydrogel beads comprise polyacrylamide cross-linked with bisacrylcystamine.

22. The method of claim 21, wherein swelling is induced in the hydrogel beads by contacting the hydrogel beads with a disulfide bond reducing agent.

23. The method of claim 1, wherein the hydrogel beads comprise polyacrylamide formed by polymerizing acrylamide and a photocleavable cross-linker.

24. The method of claim 21, wherein the photocleavable cross-linker is o-nitrobenzyl (o-NB) diacrylate.

25. The method of claim 23, wherein swelling is induced in the hydrogel beads by contact light cleavage of the photocleavable cross-linker.

26. A plurality of hollow nanovials comprising blocked openings that prevent diffusion of cells and released cell products, wherein at least some of the hollow nanovials contain one or more cells and the openings are blocked by hydrogel beads that occlude the openings.

27. The plurality of hollow nanovials of claim 26, wherein the blocked opening is formed by introducing one or more hydrogel beads into the opening and then inducing the hydrogel beads to swell such that the hydrogel beads block the opening.

28. The plurality of hollow nanovials of claim 26, wherein the hollow nanovials comprise dextran and polyethylene glycol (PEG).

29. The plurality of hollow nanovials of claim 26, wherein the hydrogel beads comprise polyacrylamide, polystyrene, silica, polymethyl methacrylate, alginate, polyethylene glycol (PEG), nylon, or agarose.

30. The plurality of hollow nanovials of claim 26, wherein a majority of the plurality of hollow nanovials contain one or zero cells.

31. The plurality of hollow nanovials of claim 26, wherein an individual hydrogel bead comprises a cloned copy of an oligonucleotide, and the oligonucleotide has a barcode sequence, and wherein the barcode sequence attached to an individual hydrogel bead is unique such that the barcode distinguishes the hydrogel bead from other hydrogel beads.

32. The plurality of hollow nanovials of claim 31, wherein the barcode sequences are attached to individual hydrogel beads via click chemistry.

33. The plurality of hollow nanovials of claim 26, wherein the oligonucleotide has a barcode sequence, wherein an individual nanovial contains a cloned copy of an oligonucleotide, and the oligonucleotide has a barcode sequence, and wherein the barcode sequence attached to an individual nanovial is unique such that the barcode distinguishes the nanovial from other nanovials.

34. The plurality of hollow nanovials of claim 33, wherein the barcode sequences are attached to individual nanovials via click chemistry.

35. The plurality of hollow nanovials of any one of claims 26 to 33, wherein the hydrogel beads comprise polyacrylamide cross-linked with bisacryloylcystamine.

36. A plurality of hollow nanovials according to any one of claims 26 to 33, wherein the hydrogel beads comprise polyacrylamide formed by polymerizing acrylamide and a photocleavable cross-linker.

37. The plurality of hollow nanovials of claim 21, wherein the photocleavable cross-linker is o-nitrobenzyl (o-NB) diacrylate.

38. A method for detecting cellular proteins in a hollow nanovial or microwell, the method comprising: Loading cells into openings in hollow nanovials or microwells; inserting one or more hydrogel beads into the opening, thereby blocking the opening and preventing the cells from diffusing from the opening; and Detection of secreted cellular proteins in blocked nanovials.

39. A method according to claim 38, wherein the nanovial or microwell contains a binding agent that specifically binds to one or more secreted cellular proteins, and the secreted cellular proteins bind to the binding agent, and a second binding agent specific for the secreted cellular proteins diffuses into the blocked nanovial or microwell to specifically detect the secreted cellular proteins bound in the nanovial or microwell.

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