Mechanisms in pairing cells and micro particles for applications in single cell investigations

Abstract

Cell-binding agents linked to beads are used to attach the beads to cells that are subsequently partitions. Methods, reaction mixtures, and kits as well as other aspects are provided.

Description

PATENTAttorney Docket No. 094868-1514265-122810PCClient .Ref. No. BR.Pl) 1298- WOMECHANISMS IN PAIRING CELLS AND MICRO PARTICLES FOR APPLICATIONS IN SINGLE CELL INVESTIGATIONSCROSS-REFERENCES TO RELATED APPLICATIONS10001] The present application claims priority to US Provisional Appln. 63 / 687,148, filed August 26, 2024, which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION 100021 Beads conjugated to oligonucleotides are used in microfluidic detection applications such as high-throughput sequencing having many different partitions (e.g., droplets). The beads for example deliver many copies of the same oligonucleotide to a partition. In order to uniquely identify each partition, the oligonucleotide copies linked to a bead will have barcode sequences unique or nearly unique for the bead to which the oligonucleotides are linked. However, if one wants to ensure that partitions have only one bead and thus are uniquely labeled by the barcode, bead concentrations are typically adjusted so that only about I out of 10 partitions are occupied by a bead. This results in low utilization of the partitions and increases the amount of sample and reagents that is needed for detection of samples.Increasing bead concentrations would result in higher partition occupancy and greater utilization of partitions. The amount of sample and reagents that are needed for detection of samples would be decreased if Poisson distributions of beads and cells could be altered.BRIEF SUMMARY OF THE INVENTION|0003| Cells can be compartmentalized into a droplet, microwell, or another compartment after being bound to a microparticle. As described herein, binding the cell to the microparticle can be facilitated by various biological materials including but not limited to cholesterol, antibodies, lectins, peptides, etc. The connection of the bead to the binding molecule can be through various polymers including ethylene glycol polymers of varying lengths, such as but not limited to, triethylene glycol, hexaethylene glycol, and nonethylene glycol, oligonucleotides, or other polymers. The pairing of the cell with the beads minimizes loss of cells, and therefore data. For example, instead of different Poisson Distributions for beads and cells into partitions, a single Poisson distribution occurs for bead / cell pairs. Beads1TOWNSEND 78761584 1containing a cell-binding agent are mixed together with cells in an aqueous solution. Before, during or after introduction into partitions, the bead-cell pairs can be mixed with reagents to facilitate single cell assays, which can include, but are not limited to, RNAseq or Assay for Transposa.se Accessible Chromatin assays (ATAC). In some embodiments, a reaction mixture is added to the cell-bead pairs and then compartmentalization into partitions can occur to create partitions that contain single cells. The compartments can be treated as needed for the specific assays as desired, including for example barcoding cell nucleic acids w ith a partition-specific barcode. Next, the products of interes t can be recovered for downstream analysis such as next generation sequencing. The methods described here are useful for sequencing any type of cells, however it is particularly useful far detecting and sequenc ing rare cel ls in a population. Examples of rare cells can include, but are not limited to, circulating tumor cells in a human’s blood.|0004] In some embodiments, a method of forming partitions containing a hydrogel bead and a cell or forming a cross-linked hydrogel surrounding physically-separated cell / hydrogel bead pairs is provided. In some embodiments, the method comprises providing a mixture of cells and hydrogel beads, wherein the hydrogel beads are linked to a plurality of oligonucleotides, wherein a cell-binding agent is linked to the hydrogel beads via a linker comprising a non-nucleic acid linker or wherein the cell-binding agent is linked to an oligonucleotide that anneals to one of the plurality of oligonucleotides linked to the hydrogel bead, wherein the cell-binding agent binds to a cell to form cell / hydrogel bead pairs such that some cells are linked to the hydrogel beads via the cell-binding agent; and introducing the cell / hydrogel bead pairs into partitions or separating the cell / hydrogel bead pairs in a cross-linked hydrogel surrounding the cell / hydrogel bead pairs, thereby forming partitions containing a hydrogel bead and a cell or a cross-linked hydrogel surrounding physically-separated cell / hydrogel bead pairs.

[0005] In some embodiments, the linker further comprises a nucleic acid. In some embodiments, the non-nucleic acid linker comprises ethylene glycol, propanediol-3-succinic acid, polyethylene glycol, abasic furan or a tetrahydrofuaran derivative mimicking art abasic site, or a polyandiol. In some embodiments, the ethylene glycol is triethylene glycol, hexaethylene glycol, or noneihylene glycol.

[0006] In some embodiments, the non-nucleic acid linker is at least 2, 4, 6, 8, 10, 1.2, 14,16, 18, 20, 22. 24, 26, 28, 30, 32, 34, or 36, (e.g., 2-36) carbons In length.TOWNSEND 7S7MSS4 i[0OO7| hi some embodiments, the method further comprises, after the providing and before the introducing, removing from the mixture (a) at least some hydrogels beads that do not bind to the cells., (b) at least some cells that do not bind to the hydrogels beads, or (c) both (a) and (b).[0008| In some embodiments, the cell-binding agent is a cholesterol moiety, a lipid moiety, a cell-adhesion polypeptide, a lectin or an antibody that binds to the cell. In some embodiments, the antibody is biotinylated and the hydrogel bead is linked to avidin or streptavidin.[0009| In some embodiments, the cell-binding agent is covalently-linked to the hydrogel bead.|0010| In some embodiments, the cell-binding agent is linked to one or more oligonucleotide from the plurality of oligonucleotides, wherein the one or more oligonucleotide is linked to the cell-binding agent via the non-nucleic acid linker.100111 In some embodiments, the cell-binding agent is linked to an oligonucleotide that anneals to one of the plurality of oligonucleotides linked to the hsdioge! bead.|O01.21 hi some embodiments, at least a majority of the plurality of oligonucleotides linked to the hydrogel beads comprise a barcode sequence unique for the bead to which the oligonucleotides are linked.[06131 In some embodiments, the method further comprises lysing the cell in the partitions. In some embodiments, the cells are lysed with a detergent. In some embodiments, the detergent is present in or introduced Into the partition. In some embodiments, the detergent is selected from the group consisting of Tween-20, Triton- X and NP40.|0014| In some embodiments, the hydrogel beads comprise a magnetic or paramagnetic particle.[0615'| In some embodiments, the partitions are microwells or aqueous droplets in an emulsion.|00l6] A lso provided is hydrogel bead linked to a plurality of oligonucleotides, wherein a cell-binding agent is linked to the hydrogel beads via a linker comprising a non-nucleic acid linker or wherein the cell-binding agent is linked to an oligonucleotide that anneals to one of the plurality of oligonucleotides linked to the hydrogel bead. In some embodiments, the3TO WE SEND 7S76HS4 llinker further comprises a nucleic acid, in some embodiments, the non-nucleic acid linker comprises ethylene glycol, propanediol-3 -succinic acid, polyethylene glycol, abasic furan or a teUnhydrofuaran derivative mimicking an abasic site, or a polyandiol. In some embodiments, the ethylene glycol is triethylene glycol or hexaethylene glycol or nonethylene glycol. In some embodiments, the non-nucleic acid linker is at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36, (e.g., 2-36) carbons in length. In some embodiments, the cell -binding agent is a cholesterol moiety, a lipid moiety, a cell-adhesion polypeptide, a lectin or an antibody that binds to the cell. In some embodiments, the antibody is biotinylated and the hydrogel bead is linked to avidin or streptavidin. In some embodiments, the cellbinding agent is covalently-linked to the hydrogel bead. In some embodiments, the cellbinding agent is linked to one or more oligonucleotide from the pl urality of oligonucleotides. In some embodiments, the cell-binding agent is linked to an oligonucleotide that anneals to one of the plurality of ol igonucleotides linked to the hydrogel bead. In some embodiments, at least a majority of the plurali ty of oligonucleotides linked to the hydrogel beads comprise a barcode sequence unique for the bead to which the oligonucleotides are linked.

[0017] Also provided is a reaction mixture comprising a cell and the hydrogel bead as described above or elsewhere herein,|'0018] Also provided is a kit comprising one or more hydrogel bead as described above or elsewhere herein. In some embodiments, the kit further comprises a detergent. In some embodiments, the detergent is selected from the group consisting of wherein the detergent is selected from the group consisting of Tween-20, Triton- X and N P40.

[0019] Also provided is a surface comprising discrete locations linked via a linker cornprising a non-nucleic acid linker to a cell-binding agent or wherein the cell-binding agent is linked to an oligonucleotide that, anneals to an oligonucleotide linked to the surface. In some embodiments, the linker further comprises a nucleic acid. In some embodiments, the non-nucleic acid linker comprises ethylene glycol, propanediol-3-succinic acid, polyethylene glycol, abasic furan or a tetrahydrofuaran derivative mimicking an abasic site, or a polyandiol. In some embodiments, the ethylene glycol is triethylene glycol, hexaethylene glycol or nonethylene glycol. In some embodiments, the non-nucleic acid linker is at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36, (e.g., 2-36) carbons in length. In some embodiments, the surface further comprises cells l inked to cel l -bi ndi ng agents. In some embodiments, the surface further comprises a layer of cross-linked hydrogel4TOWNSEND 7S76HS4 1surrounding the cells. In some embodiments, the cell-binding agent is a cholesterol moiety, a lipid moiety, a cell-adhesion polypeptide, a lectin or an antibody that binds to the cell. In some embodiments, the oligonucleotides comprise a barcode sequence unique for the discrete location to which the oligonucleotides are linked.BRIEF DESCRIPTION OF THE DRAWINGS[0020 J FIG . 1 depicts a bead attachment schematic. Polymer beads containing engineered cell-binding (BIND) agents can be mixed with cells to anchor the cell-binding agentcontaining beads to the cells. Once the pairing of cell and bead is created, the pair can be compartmentalized to facilitate downstream single ceil assays. The cell-binding agents can include cholesterol, antibodies, lectins, lipid, etc. The molecules linking the bead to the cellbinding agents can include oligonucleotides, ethylene glycol polymers of varying lengths such as but not limited to triethylene glycol, hexaethylene glycol, and nonethylene glycol carbon spacers, and polymers.|'002.tl FIG. 2 depicts cell-binding agent (BIND) motif schematics. Polymer beads containing engineered cell-binding agents can contain cholesterol linked to an oligonucleotide. As depicted, the cholesterol is added to the oligonucleotide with a triethylene glycol polymer so that there is a carbon linker between the oligonucleotide and the cholesterol. Adding additional ethylene glycol monomers creating longer spacers including hexaethylene glycol and nonethylene glycol spacers is also useful. Due to the nature of single cell assays, the polymer bead oligonucleotide is single-stranded and this allows secondary structures stabilized by nucleic acid complementation. This secondary structure formed by single-stranded oligonucleotides without a further spacer inhibits effective interactions of BIND molecules outside of the polymer bead matrix.10022] FIG. 3 depicts percent of cells co-localizing with beads as described in Example I . Beads with Cholesterol bind to cells throughout compartmentalization in oil. Beads and cells were paired in a standard PBS solution to pair them. A standard RT reaction without detergent was mixed into the solution before compartmentalization using oil. The emulsion was incubated for I hour 1 hour at 37 C. Next the emulsion was imaged on a microscope and the cells and bead counted to determine if the droplet compartment contained a cell and a bead. Cells with beads not containing cholesterol were detected in droplets with beads about 40 % of the cells. Adding with cholesterol wi th increasing carbon spacers increased cells associated with beads to about 80 % of the cells.5TOWXSEND 7S76HS4 1|0G23| FIG. 4 depicts GAPDH expression measured in cells receiving different treatments as described in Example 2. Beads with cholesterol bind to cells and can capture and synthesis GAPDH. Beads and cells were paired in a standard PBS solution to pair them. A standard RT reaction with detergent was mixed into the solution before compartmentalization, Next the emulsion was incubated for 1 hour at 37 C to promote cellular cDNA synthesis. After the cDNA synthesis step, the emulsion was broken and the quantification of GAPDH cDNA was quantified by ddPCR. The cholesterol containing beads produced more GAPDH copies in both the Igepal and Tween20 detergents.1'0024] FIG. 5A-B depicts examples of oligonucleotides that anneal to link hydrogel beads to a cell-binding agent. Beads containing an oligonucleotide that is complementary to an oligonucleotide containing the bind molecule are shown. In FIG. SA, the beads are annealed to the oligonucleotide containing the cell-binding agent (BIND) and then mixed with cells to create the bead cell pair. In FIG. SB, oligonucleotides containing the bind molecule are mixed with cells first and then beads containing the complement to the oltgomicieotide to the bind oligonucleotide are added.DEFINITIONS j0O25| Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook e / oh MOLECULAR CLONING' A LABORATORY MANUAL, 2d ed. ( 1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference), which are provided throughout this document|0026| A nucleic acid, or portion thereof ^hybridizes” to another nucleic acid under conditions such that non-specific hybridization is minimal at a defined temperature in a physiological buffer. In some cases, a nucleic acid, or portion thereof, hybridizes to a conserved sequence shared among a group of target nucleic acids. In some cases, a primer, or portion thereof can hybridize to a primer binding site if there are at least about 6, 8, 10,6TOWXSEND 7S76ISS4 i12, 14, 16, or 18 contiguous complementary nucleotides, including ‘‘universal” nucleotides that are complementary to more than one nucleotide partner. Alternatively, a printer, or portion thereof, can hybridize to a primer binding site if there are fewer than 1 or 2 complementarity mismatches over at least about 12, 14, 16, or 18 contiguous complementary nucleotides. In some embodiments, the defined temperature at which specific hybridization occurs is room temperature. In some embodiments, the defined temperature at which specific hybridization occurs is higher than room temperature. In some embodiments, the defined temperature at which specific hybridization occurs is at least about 37, 40, 42, 45, 50. 55, 60, 65, 70, 75, or 80°C.

[0027] As used herein, “nucleic acid” refers to DNA, RNA, single-stranded, double- stranded, or more highly aggregated hybridization motifs, and any chemical modifications thereof Modifications include, but are not limited to, those providing chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, points of attachment and fimctionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, peptide nucleic acids (PNAs), phosphodi ester group modifications ( e.g., phosphoroth ioates, methyl phosphonates), 2’-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines. substitution of 4-thiouridine, substitution of 5-brorno or 5-iodo-tuacil: backbone modifications, methyiations, unusual base-pairing combinations such as the isobases, isocytidine and isoguanidine and the like. Nucleic acids can also include non-natural bases, such as, for example, nitroindole, Modifications can also inchide 3’ and 5’ modifications including but not limited to capping with a fluorophore (e.g., quantum dot) or another moiety.|0028] As used herein, the term “partitioning” or “partitioned” refers to separating a sample into a plurality of portions, or “partitions.” Partitions are generally physical, such that a sample in one partition does not, or does not substantially, mix with a sample in an adjacent partition. Partitions can be solid or fluid. In some embodiments, a partition is a solid partition, e.g., a microchannel or microwell. In some embodiments, a partition is a fluid partition, e.g., 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 that is surrounded by an immiscible carrier fluid (e.g., oil). Exemplary array of wells and well descriptions can be found for example .in U.S. Patent No. 9,103,754 and 10,391 ,493, The array of wells (set of nanowells, microwells, wells) can7TOWNSEND 7S76HS4 ifunction to capture the solid supports, optionally in addressable, known locations. As such, the array of wel ls can be configured to facilitate bead capture in at least one of a single-soli d support format or optionally in small groups of solid supports. Exemplary microwell arrays and methods of delivery of beads to the micro wells and analysis thereof is described in, e.g., PCT / US2021 / 034152.|00291 The term “bead" refers to any solid support that can be In a. partition, e.g., a small particle or other solid support. Exemplary beads can include hydrogel beads, to some cases, the hydrogel is inform. In some cases, the hydrogel is inge / form. An exemplary hydrogel is an agarose hydrogel. Other hydrogels include, but are not limited to, those described in, e.g. , U.S. Patent Nos. 4,438,258; 6,534,083; 8,008,476; 8,329,763; U.S. Patent Appl, Nos. 2002 / 0,009,591 ; 2013 / 0,022,569; 2013 / 0,034,592; and International Patent Publication Nos. WO / 1997 / 030092; and WO / 2001 / 049240.IOO3O| As used herein, a “barcode" is a short nucleotide sequence (e.g., at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30 or more nucleotides long) that identifies a molecule to which it is conjugated. In some embodiments, a barcode is used to identify molecules in a partition or discrete location on a surface. Such a partition-specific barcode (or multiple different partition-specific barcodes) can be unique for that partition or discrete location as compared to barcodes present in other partitions. Partition-specific barcodes can be delivered to partitions for example linked to beads that are delivered to a partition, after which barcoding oligonucleotides are released from the beads and linked to a nucleic acid in the partitions, e.g., from a single cell in the same partition. Thus, nucleic acid from each cell can be distinguished from nucleic add of other cells due to the unique ‘'cellular barcode,” In some embodiments, a barcode is present on oligonucleotides conjugated to a particle, wherein the “particle barcode” or “bead barcode” is shared by (e.g. , identical or substantially identical amongst) all, or substantially all, of the oligonucleotides conjugated to that particle or bead. When delivered to a partition, a bead barcode can act as a “partition-specific barcode.” In some embodiments, the barcode is discontinuous.|0031] The length of the underlying barcode sequence determines how many unique samples can be differentiated. For example, a 1 -nucleotide barcode can differentiate 4, or fewer depending on degeneracy, different partitions; a 4-nucleotide barcode can differentiate 4* or 256 partitions or less; a 6-nucleotide barcode can differentiate 4096 different partitions or less; and an 8 nucleotide barcode can index 65,536 different partitions or less.8TOWXSEND 7S76HS4 1[0032| “Clonal” copies of a polynucleotide mean the copies are identical in sequence. In some embodiments, there are at least 100, 1000, 104or more clonal copies of oligonucleotides in linked to a bead.DETAILED DESCRIPTION OF THE INVENTION|0033| The inventors have discovered that hydrogel beads carrying barcoding oligonucleotides can be localized to cells by including a cell-binding agent with the hydrogel beads. The cell-binding agents can localize the beads to the cells. The resulting cell / bead pairs (cells bound to one ar more bead via the cell binding agents linked to the beads) can subsequently be introduced into partitions. In some embodiments, this will result in a higher concentration of partitions containing a cell and at least one barcoding bead compared to what occurs based on Poisson distribution if independently delivered cells and beads. As an alternative to beads, the inventors have also determined that a surface with cell-binding agents bound to discrete locations on the surface can be used to localize cells to the locations, and if desired, the cells can then be coated with a cross-linked hydrogel that seals the cells to the locations while allowing for diffusion of reagents to the cells for analysis.10034] Cells can be compartmentalized into a droplet, microwell, or another compartment after being bound to a microparticle. Binding the cell to the microparticle can be facilitated by various biological materials including but not limited to cholesterol, antibodies, lectins, peptides, etc. The connection of the bead to the binding molecule can be through various polymers including triethylene glycol, oligonucleotides, or other polymers. The pairing of the cel l wi th the beads allows minimize loss of cells, and therefore data. Beads containing an attachment molecule can be mixed together with cells in an aqueous solution. The bead-cell pairs can be mixed with reagents to facilitate single cell assays including RNAseq, Assay for Transposase Accessible Chromatin assays (A.TAC), etc. Once the reaction mixture is added to the cell-bead pairs, compartmentalization can occur to create single cell results. The compartments can be treated as needed for the specific assays. Next, the products of interest can be recovered for downstream analysis such as next generation sequencing.|0035| As shown in the examples, it has further been discovered tha t inclusion of a non- uucleic linker between the hydrogel bead and the cell-binding agent significantly increases the abi lity of the cell-binding agent to bind to a cell. In some embodiments, the linker further comprises a nucleic acid in addition to the non-nncleic linker. Without intending to limit the9TOWXSEND 7S76HS4 iscope of the invention, it is believed that the non-nucleic acid portion of the linker prevents interaction with itself as well as promotes the cell-binding agent interacting with the cell. Examples of non-nucleic acid linker components can include, for example, ethylene glycol (for example, but not limited to, triethylene glycol, hexaethylene glycol, nonethylene glycol or longer triethylene glycol polymers), propanedioI-3-succinic acid, polyethylene glycol, abasic furan or a tetrahydrofoaran derivative mimicking an abasic site, or a polyandiol. The non-nucleic acid linker can be 2 carbons or more in length, for example in some embodiments, the non-nucleic acid linker is at least 2, 4, 6, 8, 10, 1'2, 14, 16, 18, 20. 22, 24, 26, 28, 30, 32, 34, or 36 (e.g,, 2-36, 2-20. etc.) carbons in length. The number of carbons refer to the length of the non-nucleic acid linker backbone and not side chains. The backbone can optionally comprise other atoms as well. For example, when oxygen can also be present in the backbone, for example for every two carbons in the backbone one oxygen can be present in the backbone, which is exemplified in the examples.100361 In aspects described herein, beads (for example but not limited to, hydrogel beads) linked to cell-binding agents are provided. Any bead of usefol size and composition for delivery to partitions can be used. The panicle or bead can be any particle or bead having a solid support surface. Solid supports suitable for particles include controlled pore glass (CPG)(available from Glen Research, Sterling, Va.), oxalyl-controlled pore glass (See, e.g. , Alni, el ak, Nucleic Acids Research 1991, 19, 1527), TeiitaGel Support — an aminopolyethyleneglycol derivatized support (&e. e,,g. , Wright, et al... Tetrahedron Letters 1993, 34, 3373), polystyrene, Poros (a copolymer of polystyrene / divinylbenzene), or reversibly cross-linked acrylamide. Many other solid supports are commercially available and amenable to the methods described herein. In some embodiments, the bead material is a polystyrene resin or poly(methyl methacrylate) (PMMA '), The bead material can be metal,J0037| In some embodiments, the particle or bead comprises hydrogel or another similar composition. In some cases, the hydrogel is in xol form. In some cases, the hydrogel is in gel form. An exemplary hydrogel is an agarose hydrogel. Other hydrogels include, but are not limited to, those described in, e.g.. U.S. Patent Nos. 4,438,258; 6,534,083; 8,008,476;8,329,763; U.S. Patent Appi. Nos. 20020009591 ; 20130022569; 20130034592; and International Patent Publication Nos. WO 1997030092; and W02001049240. Additional compositions and methods for making and using hydrogels, such as barcoded hydrogels, include those described in, e.g., Klein et aL, Cell, 2015 May 21 ;I61(5):1 187-201. Beads can be magnetic or paramagnetic, or have a magnetic or paramagnetic core, as desired.10TOWN SEND 7S76KS4 i[0038| The solid support surface of the bead can be modified to include a linker for attaching barcode oligonucleotides, 'Hie linkers may comprise a cleavable moiety, which may be cleaved before reverse transcription is performed in the partitions. Non-limiting examples of cleavable moieties include a disulfide bond, a dioxyuridme moiety, and a restriction enzyme recognition site. The cleavable sequence can be any cleavable sequence that can be targeted enzymatically or otherwise while leaving the rest of the nucleic sequences in the mixture intact. In some embodiments, the cleavable sequence comprises one or more uracils. For example, the cleavable sequence can include 1, 2. 3, 4 or more uracils, which can be contiguous. Uracils can be selectively removed and the backbone cleaved (nicked) by contacting with uracil DNA glycosylase and endonuclease VIII, which excises the one or more uracil. Uracil DNA glycosylase and endonuclease VIII is available commercially, for example from New England Biolabs as ‘‘USER***’ (Uracil-Specific Excision Reagent). In some embodiments, the cleavable sequence comprises one or more ribonucleotide(s). For example, the cleavable sequence can include 1 , 2, 3. 4 or more ribonucleotides, which can be contiguous. This allows one to use an enzyme that selectively Cleaves ribonucleotides and does not substantially cleave deoxribonucleotides. For example, in some embodiments, RNAseH is used to specifically cleave at a ribonucleotide in the cleavable sequence. Preferably, a restriction enzyme is selected such that its recognition and / or cleavage site only occurs in the cleavable sequence and does not occur elsewhere in the oligonucleotides in the mixture.(0039j Oligonucleotides can be linked to beads as desired. Methods of linking oligonucleotides to beads are described in, e.g., WO 2015, 200541. In some embodiments, the oligonucleotide configured to link a hydrogel bead to the barcode is covalently linked to the hydrogel, Numerous methods for covalently linking an oligonucleotide to one or more hydrogel matrices are known in the art. As but one example, aldehyde derivatized agarose can be covalently linked to a 5 ’-amine group of a synthetic oligonucleotide.[0040J In some embodiments, the hydrogel beads are covalently-linked to the cell-binding agents, optionally via a non-nucleic acid linker as described herein. The cell-binding agent(s) can be covalently linked directly to the hydrogel beads, or the agents can be linked via a spacer or other linker, For example, the inventors have found that improved cell binding can occur if a nou-nucleic add spacer links the agents to the beads. In some embodiments, the cell-binding agent is linked to an oligonucleotide that is linked to the bead. For example, in some embodiments, the cell-binding agent is linked to the 3’ end of an ol igonucleotide that is 11TOWNSEND 755761584 1linked io the bead, optionally also comprising a further non-nucleic acid spacer molecule between the 3’ end and the cell-binding agent. Exemplary spacer molecules to link the cellbinding agent to the bead can include, but are not limited to, carbon spacers, e.g, C3, C6, C9, Cl 2, Cl 5, or CIS spacers, though other types of spacers can also be used as desired. Exemplary spacer molecules can include, for example, triethylene glycol spacers, for example but not limited to triethyelene glycol, hexaethylene glycol, and nonethylene glycol. See, e.g., O’Dea, et M , Current Protocols in Nucleic Acid Chemistry 01 May 2001 doi.org / 10.1002 / 0471142700.nc0503s00. In other embodiments, the non-nucleic acid spacer can be propanediol-3-succmic acid, polyethylene glycol, abasic furan or a tetrahydrofuaran derivati ve mimicking an abasi c site , or a polyandiol. Linking of polymers to oligonucleotides can be performed as desired. In some embodiments, amidite or phosphoamidite chemistry approaches can be used.|O941] In embodiments in which the cell-binding agent is linked to oligonucleotides (optionally further comprising a non-nucleic acid spacer as described herein) that are linked to the bead, the oligonucleotides linked to the cell-binding agents can be (i) a subset of the barcoding oligonucleotides on the bead, or (it) a different oligonucleotide (having a different sequence than the barcoding oligonucleotides). For example, in some embodiments, the bead is linked to 100s or 1000s of clonal (clonal except possibly for a UM1 sequence) copies of barcoding oligonucleotides, i.e, oligonucleotides having a barcode sequence unique for the bead, and a traction of those barcoding oligonucleotides are linked to a cell-binding agent. The fraction can be varied as desired, but in some embodiments, is less than 20%, 15%, 10%, 5%, 1% or less of the oligonucleotides linked to the bead, as the cell-binding agents can block usage of the oligonucleotides for downstream molecular reactions. In some embodiments, the hydrogel beads have a nucleic acid to which oligonucleotides can be ligated. In these embodiments, a mixture of oligonucleotide and 3’ linked cell binding agent / o'ligonucleotide can be ligated to the bead in bulk m ix ture, wherein the ratio of “plain” (lacking a cell-binding agent) oligonucleotide to 3’ linked cell binding agent / oligonucleotide determines the ratio on the bead. If the linker between the cell-binding agent and the bead is not an oligonucleotide, a reactive moiety (e.g, an aery lie group) can be added on one end of the linker and the cellbinding agent on the other end of the linker so dial during polymeri zation of the polyacrylamide (e.g, forming the bead), the acrylic group on the linker is incorporated into the polyacrylamide chain and the matrix of the bead,12TOWXSEND 7S76HS4 i[0042| hi some embodiments, the hydrogel beads are non-cevaleufly-linked to the cell- binding agents. Any non-covalent linkage of the hydrogel beads to the cell-binding agents is contemplated. In some embodiments, the hydrogel bead is covalently or non-covalently linked to a first oligonucleotide (which may or may not be a barcoding oligonucleotide) and the cell-binding agent is covalently or non-covalently linked to a second oligonucleotide, wherein the first oligonucleotide and the second oligonucleotide have reverse complementary sequences, allowing the two oligonucleotides to anneal, thereby linking the hydrogel bead to the cell-binding agent. See, e.g., FIG. 5. Other non-covalent binding options include but are not limited to streptavidin-biotm pairs, or antibody-epitope pair binding, in which one of the pairs is linked to the hydrogel bead and the other of the pair is linked to the cell-binding agent.|0043| Any cell-binding agent can be used that has affinity for the desired cell. In many embodiments., the cell-binding agent will have affinity for any cell in a mixture. For example, a lipophilic agent can be used to target and embed in a cell membrane. Exemplary lipophilic agents can include but are not limited to cholesterol and derivatives thereof or lipids. When mixed with cells, a lipophilic agent linked to a hydrogel bead will pair the bead to the cell by embedding into the cell membrane. The cholesterol or lipid-derivative cellbinding agents embed into the cell or nuclear membrane. Protocols for using lipid-derived or cholesterol or derivatives to attach to cells are described in, e.g,, McGinnis, e / a / ., Admre MerfexA 16;619-626 (2019); ’Weber ef al, Biomacromolecul&i 15:4621-4626 (2014). In some embodiments, the cell-binding agents comprise a spacer between the lipid or cholesterol moiety and the oligonucleotide and / or the hydrogel bead. The lipid moiety, in some embodiments, includes an ethylene glyco! chain. Exemplary ethylene glycol molecules comprise two carbons and one oxygen .in. the backbone of the linker and polymers of triethylene glycol can be used as the linker, for example such that 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more contiguous triethylene glycol molecules are in the linker. The oligonucleotides may be any length, for example 10-80 nucleotides long, e.g., 10-60, 10-40, 20-80, 20-60, 20-40, 40-60, 40-80, 50-80, 50-80, or 60-80 nucleotides long.(0044| In some embodiments, the cell-binding agent is a cell-adhesion polypeptide. Exemplary cell-adhesion polypeptides include but are not limited to those that comprise the “RGD” (Arg-GIy-Asp) amino acid sequence that is recognized by a number of cellular integrins, and thus allow for binding of the cell adhesion polypeptide to cells via integrins embedded in the cell membrane. One example of RGD proteins is fibronectin though many 13TOWNSEND 7S7M5S4 iothers can also be used. See, e.g.sBellis, Biomaieria / s 2011 Jun; 32(18): 4205-4210;Ruoshlati, Anmi Bev Cel / .Dev Biol. 1996: 12:697-715. In another example the cell-adhesion polypeptides comprise heparin.e.g., Liu el al., Cell Biology and Metabolism, Vol. 273, Issue 16, P9718-9726, April 1998. In another embodiment, the cell-adhesion polypeptides comprise laminin. .See, e.g., Hozumi, el al, Biomateriab\ Volume 33, Issue 17, June 2012, Pages 4241-4250. In general, cell-adhesion proteins will bind to multiple cell types, assuming the cells have an integrin protein that binds to the cell-adhesion protein.|O04S] In some embodiments, the cell-binding agent is a lectin or other polysaccharide- binding agent that can bind to a sugar or polysaccharide on the outside of a cell.10046] In some embodiments, the cell-binding agent is an antibody or other protein (e.g.. a receptor ligand) drat binds to one or more targets on a cell. An antibody or receptor ligand protein can be used to se lecti vely bind to specific target cells in a mixture of different cel ls in which some cells comprise the target epitope or receptor while other cells do not, allowing for enriched targeting of certain cells. Methods of linking antibodies to oligonucleotides can include those described in Jones, ez oZ., Scientific Beporls volume I I, Article number: 23844 (2021).|0047| As noted above, in some embodiments, the hydrogel beads also are linked to barcoding oligonucleotides (e.g., a plurali ty oof clonal oligonucleotides) to be deli vered wi th the cells into partitions. The barcoding oligonucleotides can comprise, for example, 5 ’-3’: a first universal adapter sequence, a bead-specific barcode sequence and a 3’ end that targets desired sample nucleic acids. For example, the barcoding oligonucleotides can be used as RT primers (e.g., having a poly-T, gene-specific, or random 35end sequence) by annealing to RNA. in a partition to form cDNAs that include the bead-specific barcode sequence, or to primer amplification (e.g., PCR) reactions using cellular RNA or DNA, which has optionally been treated with a transposon (e.g., tagmentase) to fragment the nucleic acids and add common adapter sequences. Additional barcodes, such as but not limited to. unique molecule identifiers (UMIs) or sample-specific barcodes can also be included in the oligonucleotide sequence. Note the barcoding oligonucleotides can be clonal, e.g., identical, other than the UMI sequence.|0048] Cells and hydrogel beads linked to the cell-binding agent can be mixed as desired such that cells are bound by the cell-binding agents. In some embodiments, for example, cells and hydrogel beads linked to cell-binding agents are vortexed, e.g., in bulk, mixed by14TOWXSEND 7S7SHS4 1microfluidics mixing or otherwise mixed under conditions in which the cell-binding agents bind to the cells. In some embodiments, two aqueous solutions are mixed so beads and cells are mixed one for one (one cell and one (or a few such as no mor than five) bead pairing), which can be achieved for example using microfluidics. In embodiments in which the cellbinding agent is non-covalently linked to the hydrogel bead (e.g.. via annealing oligonucleotides as described above), in some embodiments, the cell binding agent and hydrogel beads are mixed initially so that the cell-binding agent is linked to the hydrogel beads followed by subsequent mixing with cells, hi other embodiments, other order of events can take place, for example all three components can be mixed together, allowing the cellbinding agent to bind to the cells and the hydrogel beads. The ratio of hydrogel beads, cellbinding agents and cells can be selected to optimize the number and proportion of cells bound to hydrogel beads as well as optimizing the average number of hydrogel beads bound per cell. For example, in some embodiments, the ratios are selected such that a high (greater than 50, 70, or 90%) proportion of the ce lls are bound by at least one hydrogel bead. In some embodiments, the number of hydrogel beads in the reaction mixture is higher than the number of cells. For example, the number of hydrogel beads can be at least 2 , 4, 6, 8, 10 (e.g., 2-20) times the number of cells in the reaction mixture. This- can be helpful for example in improving the portion of cells bound by at least one bead. Cell / hydrogel bead pairs are formed by the interaction of the cell-binding agents with the hydrogel beads and the cells. Although the term “pair” is used, it will be appreciated that in some embodiments, there will be one or more hydrogel bead associated with a cell and for the purposes of convenience, the term<kpair” is intended to encompass these embodiments unless indicated otherwise,|0049| Any type of cells (or isolated nuclei as desired, e.g., for ATAC methods) can be used according to the methods and compositions described herein. In some embodiments, the cells are mammahan, for example human cells. In some embodiments, the cells are from a biological sample. Biological samples can be obtained from any biological organism, e.g.. an animal, plant, fungus, pathogen (e,g., bacteria or virus), or any other organism. In some embodiments, the biological sample is from an animal, e.g., a mammal (e.g., a human or a non-haman primate, a cow, horse, pig, sheep, cat, dog, mouse, or rat), a bird (e.g,, chicken), or a fish. A. biological sample can be any tissue or bodily fluid obtained from the biological organism, e.g., blood, a blood fraction, or a blood product (e.g., serum, plasma, platelets, red blood cells, and the like), sputum or saliva, tissue (e.g., kidney, lung, liver, heart, brain,15TOWXSEND 7S76HS4 1nervous tissue, thyroid, eye, skeletal muscle, cartilage, or bone tissue); cultured cells, e.g,, primary cultures, explants, and transformed cells, stem cells, or cells found in stool, urine, etc.|0050| In some embodiments, the cells are fixed cells. For example in some embodiments, the cells are formalin-fixed, paraffin-embedded (FFPE) samples. In some embodiments, the cells are not fixed. In either case, in some embodiments, the cells are permeabilized to allow for entry of reagents while the cells themselves remain substantially intact. In some embodiments, once the cells or nuclei are present in partitions, the cel ls or nuclei are lysed to release cellular RNA in cells or nuclei into the partitions, allowing for the RNA to take part in a reverse transcription reaction, optionally primed from an oligonucleotide from a hydrogel bead, optionally providing a partition-specific barcode and / or other end sequences to a cDNA. In some embodiments, the cells or nuclei in the partitions have been permeabilized such that the RNA in the permeabilized cell or nuclei can diffuse from the cells or nuclei into the remaining inside portion of the partitions. Permeabilization can remove cellular membrane lipids to allow large molecules such as enzymes to enter the cell. In some embodiments, a detergent is used for permeabilization, Exemplary detergents can include, for example, Triton X-100 and NP-40 are used for permeabilization (for example, at 0.1- 0.5% (v / v, in PBS). In some embodiments, a steroidal saponin (or saraponin) is used to solubilize lipid, resulting in permeabilization.. An exemplary saraponin is Digitonin. The appropriate permeabilization reagent can be selected to be compatible with the integrity of the partition used. In some embodiments, the cells or isolated nuclei are lysed so that their contents, including DNA or RNA, are released into the partition in which the cell or nucleus is present.|0051 ] Following formation of cell / hydrogel bead pairs (e,g., by mixture of the cells and. cell-binding agents linked to hydrogel beads), optionally the mixture can be enriched for the celVhydrogel bead pairs by removing (i) at least some cells that are not paired with hydrogel beads, (ii) hydrogel beads that are not paired with cells, or (Hi) both (i) and (ii). In some embodiments, a characteristic of the hydrogel beads or the cells can be used to enrich for one of these components, thereby removing at least some other components that are unbound. As one example, if the hydrogel beads comprise a magnetic or paramagnetic core, the hy drogel beads can be isolated from unbound components by attracting the hydrogel beads and any bound components via magnetism, and allowing for rinsing or other removal of other unbound components in the mixture. In some embodiments, the cells and beads are different16TOWNSEND 7S76KS4 1sizes such that the beads, or the cells, can be removed by filtering, such that the larger of the two (beads or cells) are retained by the filter while the other is in the filtrate. In some embodiments, the cells are larger than the beads. For example, in some embodiments, the beads can be less than 10 microns, e,g., 1-10 or 1-5 microns. In some embodiments, the beads are larger than the cells. For example, in some embodiments, the beads can be more than 10 or more than 20 microns, e.g , 15-25 microns.100521 Following formation of cell / hydrogel bead pairs (and optional enrichment for the pairs as described above), the pairs can be introduced into partitions. For example, the number of pairs and partitions can be adjusted so that a majority of partitions that contain a pair contains only one pair, e.g., as commonly used in single cell sequencing applications.[0053 | Methods and compositions for partitioning are described, for example, in published patent applications WO 2010 / 036352, US 2010 / 0173394, US 2011 / 0092373, and US 2011 / 0092376. The plurality of partitions can be, for example, a plurality of emulsion droplets, or a plurality of microwells, SPCs, eie.|W54] In some embodiments, the pairs, or one or more reagents, are added during droplet formation or to the droplets after the droplets are formed. Methods and compositions for deli vering reagents to one or more partitions .include microfluidic methods as known in the art; droplet or microcapsule combining, coalescing, fusing, bursting, or degrading (e.g>, as described in U.S. 2015 / 0027,892; US 2014 / 0227,684; WO 2012 / 149,042; and WO 2014 / 028,537); droplet injection methods (e.g., as described in WO 2010 / 151 ,776); and combinations thereof|0055| The partitions can be picowells, nanowells, or microwells. The partitions can be pico-, nano-, or micro- reaction chambers, such as pico, nano, or microcapsules. The partitions can be pico-, nano-, or micro- channels. The partitions can be droplets, e.g. , emulsion droplets. In some embodiments, a droplet comprises an emulsion composition, / .<?., a mixture of immiscible fluids (e.g., water and oil). In some embodiments, a droplet is an aqueous droplet that is surrounded by an immiscible carrier fluid (e.g., oil). In some embodiments, a droplet is an oil droplet that is surrounded by an immiscible carrier fluid (e.g., an aqueous solution). In some embodiments, the droplets described herein are relatively stable and have minimal coalescence between two or more droplets. In some embodiments, less than 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of droplets generated from a sample17TOWNSEND 7S7&IS84 1coalesce with other droplets. 'The emulsions can also have limited flocculation, a process by which the dispersed phase comes out of suspension in flakes. In some cases, such stability or minimal coalescence is maintained for up to 4, 6, 8, 1.0, 12, 24, or 48 hours or more (e.g., at room temperature, or at about 0, 2, 4, 6, 8, 10, or 12 "C). In some embodiments, the droplet is formed by flowing an oil phase through an aqueous sample or reagents.|0056| The oil phase of an emulsion can comprise a fluorinated or non-fluorinated base oil which can additionally be stabilized by combination with a fluorinated surfactant such as a perfluorinated polyether or a non-fluorinated (e.g., anionic) surfactant. Exemplary oil phase compositions along these lines are described rn, e.g., PCT" WO 2020 / 247950 and US Patent Publication No. 2017 / 0175 I 79,

[0057] In some embodiments, the sample is partitioned into, or into at least, 500 partitions, 1000 partitions, 2000 partitions, 3000 partitions, 4000 partitions, 5000 partitions, 6000 partitions, 7000 partitions, 8000 partitions, 10,000 partitions, 15,000 partitions, 20,000 partitions, 30,000 partitions, 40,000 partitions, 50,000 partitions, 60,000 partitions, 70,000 partitions, 80,000 partitions, 90,000 partitions, 100,000 partitions, 200,000 partitions, 300,000 partitions, 400,000 partitions, 500,000 partitions, 600,000 partitions, 700,000 partitions, 800,000 partitions, 900,000 partitions, 1,000,000 partitions, 2,000,000 partitions, 3,000,000 partitions, 4,000,000 partitions, 5,000,000 partitions, 10,000,000 partitions, 20,000,000 partitions, 30,000,000 partitions, 40,000,000 partitions, 50,000,000 partitions, 60,000,000 partitions, 70,000,000 partitions, 80,000,000 partitions, 90,000,000 partitions, 100,000,000 partitions, 150,000,000 partitions, or 200,000,000 partitions.10058] In some embodiments, the droplets that are generated are substantially uniform in shape and / or size. For example, in some embodiments, the droplets are substantially uniform in average diameter. In some embodiments, the droplets that are generated have an average diameter of about 0.001 microns, about 0.005 microns, about 0,01 microns, about 0,05 microns, about 0.1 microns, about 0,5 microns, about 1 microns, about 5 microns, about 10 microns, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns, about 70 microns, about 80 microns, about 90 microns, about 100 microns, about 150 microns, about 200 microns, about 300 microns, about 400 microns, about 500 microns, about 600 .microns, about 700 microns, about 800 microns, about 900 microns, or about 1000 microns. In some embodiments, the droplets that are generated have an average diameter of less than about 1000 microns, less than about 900 microns, less than about 800 microns, less18TO WE SEND 7S7&IS84 ithan about 700 microns, less than about 600 microns, less than about 500 microns, less than about 400 microns, less than about 300 microns, less than about 200 microns, less than about 100 microns, less than about 50 microns, or less than about 25 microns. In some embodiments, the droplets that are generated are non-uniform in shape and / or size.

[0059] In some embodiments, the droplets that are generated are substantially uniform in volume. For example, the standard deviation of droplet volume can be less than about 1 picoliter, 5 picoliters, 10 picol iters, 100 picoliters, 1 nL, or less than about. 10 nL. In some cases, the standard deviation of droplet volume can be less than about 10-25% of the average droplet volume. In some embodiments, the droplets that are generated have an average volume of about 0.001 nL, about 0.005 nL, about 0.01 nL, about 0,02 nL, about 0,03 nL, about 0.04 nL, about 0.05 nL, about 0.06 nL, about 0.07 nL, about 0.08 nL, about 0.09 nL, about 0.1 nL, about 0.2 nL, about 0.3 nL, about 0.4 nL, about 0.5 nL, about 0.6 nL, about 0.7 nL, about 0.8 nL, about 0.9 nL, about 1 nL, about 1.5 nL, about 2 nL, about 2.5 nL, about 3 nL, about 3.5 nL, about 4 nL, about 4.5 nL, about 5 nL, about 5.5 nL, about 6 nL, about 6.5 nL, about 7 nL, about 7.5 nL, about 8 nL, about 8.5 nL, about 9 nL, about 9.5 nL, about 10 nL, about 11 nL, about 12 nL, about 13 nL, about 14 nL, about 15 nL, about 16 nL, about 17 nL, about 18 nL, about 19 nL, about 20 nL, about 25 nL, about 30 nL, about 35 nL, about 40 nL, about 45 nL, or about 50 nL.|'0060] An SPC is a capsule having a semi-permeable shell that allows for small molecules to pass through the shell while substantially retaining larger molecules, such as DNA (e.g., having at least 100 or at least 500 nucleotides), niRN A and optionally some proteins. For example WO-20231 17364 describes SPCs with a semipertneable shells comprising a gel formed from a polyampholyte and / or a polyelectrolyte, wherein the polyampholyte and / or the polyelectrolyte in the gel is covalently cross-linked. In some embodiments, as described in WO-20231 1.7364, the SPCs comprise an inner core in a liquid form, or in a hydrogel form and optionally enr iched in polyhydroxy compounds belonging to the class of polysaccharides, oligosaccharides, carbohydrates, or sugars. For example, the core can comprise a polyhydroxy compound and / or an antichaotropic agent. In other embodiments, WO- 2023099610 describes processes for manufacturing core-shell microcapsules and methods for using core-shell microcapsules to compartmentalize and optionally process biological entities and molecules. Other SPC formulations can comprise poiyfethylene glycol) diacrylate (PEGDA), for example as described in Michielin and Maerkl,Rep. 2022; 12: 21391.Bomi <7 Langmuir 2015, 31 , 6027-6034 describes yet other SPCs based on water-in-oil-19TO WN SEN D 7S7MSS4 1in-water droplets that have a middle layer composed of photocurable resin and inert oil. In some embodiments, following encapsulation of single cells into SPCs, reagents can be diffused into the beads* The SPC will be selected to have a composition to substantially retain the nucleic acids of the cells while allowing for diffusion of reagents into the SPCs.|0061| Following capture of the ceiVhydrogel beads in partitions, any downstream processing steps can be performed as desired. For example, barcoding oligonucleotide from the hydrogel beds can optionally be released from the beads and be used to barcode nucleic acids from the cell in the partition. For example, cDNAs or genomic DNA from the cells can be linked to the barcoding oligonucleotides, allowing for subsequent combination of contents of partitions while allowing, via the barcodes, to determine sequences that came from a common cell because they have a common partition barcode. In some embodiments, the cells in the partitions are lysed. In some embodiments, a detergent is present in, or added to, the partitions, in sufficient concentration to lyse the cells contained in the partitions without significantly disrupting the partitions themselves. In some embodiments, the detergent is Polysorbate 20 (e.g., Tween-20™), Ct4H22O(C3H4O)n (e.g,. Triton~X™) or nonyl phenoxypolyethoxylethanol (e.g., CAS Registry Number 9016-45-9, NP40),[0062| In other embodiments, a surface can be used to form an array of cells, For example, a surface comprising discrete locations linked to oligonucleotides linked to a cell-binding agent can be provided. The discrete locations can form an array of locations, where different locations are linked to a cell binding agent and a plurality of clonal barcoding oligonucleotides, wherein the barcodes include for example a barcode sequence unique for the location in the array.[0$63| An “array” is an ordered plurality of i tems, The term can refer to an ordered plurality of oligonucleotides, or the ordered array linked to a solid surface that is optionally planar. “Ordered” refers to known locations on the array and is not intended to indicate a particular alignment of the items, though in some embodiments the items can be in a grid.[0064| The array can be designed and generated such that clonal copies of oligonucleotides are provided in spots on the array. Each spot will have different oligonucleotides (at least differing by a barcode sequence in the oligonucleotide sequences) such that each spot’s oligonucleotides are unique for that spot, allowing one to use the sequence of the clonal oligonucleotides at the spot to identify the position of barcoding oligonucleotide, and therefore any nucleic acids linked to the oligonucleotides, at that spot. As used herein, the20TO WE SEND 7S7SHS4 iterm “spot” refers to a defined area of the array at which a particular set of clonal oligonucleotides reside. The spot size can be selected such that no more than one cell can “fit” at a spot.|0065] Methods of generating arrays of oligonucleotides with known sequences are known and can be used to generate the arrays described herein. See, for example, US2O21 / O332351 and US2020 / 0299322 or as otherwise described by for example DMA Script or TwistBiosciences. These methods, can for example, provide for spatially addressable oligonucleotides on a planar array, meaning that the oligonucleotide sequences at each spot on the array are known. In some embodiments, such planar supports have a plurality of sites comprising at least 256 sites, at least 512 sites, at. least 1024 sites, at least 5000 sites, at least10,000 sites, at least 25,000 sites, or at least 100,000 sites and as many as 10,000,000 sites.In some embodiments, the discrete site at which synthesis of spots take place each has an area in the range of from 0.25 pnr to I 000 pm2, or from 1 pm2to 1000 pm2, or from 10 pm2to 1000 pm2, or from 100 pm2to 1000 gm2. In some embodiments, the amount of polynucleotides synthesized at each spot is at least 1 O '2' final, or at least 10"'3fmol, or at leastI finol, or at least I pmol, or the amount of polynucleotide synthesized at. each spot, is in the range of from IO"2' fmol to 1 finol, or from KT2fmol to 1 finol, or from 1 fmol to 1 pmol, or from IO"*’ pmol to 10 pmol, or from IO'6pmol to 1 pmol. In some embodiments, the number of polynucleotides synthesized at each spot is in the range of from 1000 molecules to 106molecules, or from 1000 molecules to 10*2molecules, or from 1000 molecules to IO12molecules. In some embodiments, the array is on a flow cell. The array can be on any solid rigid or semi-rigid planar surface. Examples of materials composing the surface can include but are not limited to glass and plastic.|0066] In the surface embodiments, like the hydrogel bead options, the cell-binding agent can be linked to some of the barcoding oligonucleotides at spots on the surface, or on other oligonucleotides at the spots, or can otherwise be linked to the spots. In some embodiments, the cell-binding agents are linked to the surface via a non-nucleic linker as described elsewhere herein, optionally where the linker further comprises an oligonucleotide. Cells can subsequently be contacted to the surface under conditions such that indi vidual cells bind to the cell-binding agents on the surface. For example, if the surface is a flow cell, cells can be flowed across the surface.21TOWXSEND 7S76HS4 i[00671 After cells are bound at the spots on the surface, liquid form of a hydrogel can be applied to cover the cells on the surface and induced to cross-link, thereby immobilizing the different cells at their locations on the surface. In some embodiments, the hydrogel comprises alginate, agarose, polyacrylamide, chitosan, hyaluronan, dextran, collagen, fibrin, polyethylene glycol (PEG), poly(hydroxyethyl methacrylate) (polyHEMA), polyvinyl alcohol (PVA) or polycaprolactone (PCL). In some embodiments, the hydrogel comprises alginate and the crosslinking comprises contacting the hydrogel with calcium. Agarose can be crosslinked when cooled. Polyacrylamide can be polymerized with cross-linkers such as N,N'~ Bis(actyloyl)cystainine and the reaction can be initiated by contacting the hydrogel with TEMED and ammonium persulfate (APS).

[0068] After the cells are embedded in the solidified hydrogel, the barcode oligonucleotides attached to the cells can be released, e.g., by diffusing in one or more reagents that release the oligonucleotides .from the surface. Because the hydrogel is solidified around the cells, the released oligonucleotides should not diffuse very far from the membrane / hydrogel interface. Molecular reactions, which can include, but need not be limited to, cell lysis, reverse transcription, primer extension using the barcoding oligonucleotides, ligation of the barcoding oligonucleotides, or other molecular reactions can be performed while the cells are embedded in the cross-linked hydrogel. Following barcoding of the nucleic acids, the. hydrogel cross-linking can be reversed or degraded and the barcoded nucleic acids of the various cells on the surface can be combined and if desired further downstream reactions can be performed in bulk. In some embodiments, the barcoded nucleic acids are sequenced.

[0069] Barcoded nucleic acids from cells can be sequenced if desired. Sequencing platforms can be selected as desired. In some embodiments, Illumina’iM-supported sequencing methods are employed. See, e.g,, U.S. Patent Nos 11 ,029,513; US 11 ,150,179; 1 1,308,640; and 1 1 ,473,067 and citations therein. Exemplary DMA sequencing techniques include fluorescence- based sequencing methodologies (’fee, e.g., Bitten et al. Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y.; herein incorporated by reference in its entirety). In some embodiments, automated sequencing techniques understood in that art are utilized. In some embodiments, the present technology provides parallel sequencing of partitioned amplicons (PCT Publication No. WO 2006 / 0841,32, herein incorporated by reference in its entirety). In some embodiments, DNA sequencing is achieved by parallel oligonucleotide extension (fee, e.g., U.S. Pat. Nos. 5,750,341 ; and 6,306,597, both of which are herein incorporated by reference in their entireties). Additional examples of sequencing22TOWNSEND 7S76I5S4 1techniques include the Church polony technology (Mitra e / al., 2003, Analytical Biochemistry 320; 55-65; Shendure el al., 2005 Science 309, 1728-1732; and U.S. Pat. Nos. 6,432.360; 6,485,944; 6,51 1 ,803; herein incorporated by reference in their entireties), the 454 picotlter pyrosequencing technology (Margulies er of, 2005 Nature 437, 376-380; U.S. Publication No. 2005 / 0130173; herein incorporated by reference in their entireties), the Solexa single base addition technology (Bennett el al., 2005, Phannacogeno.rn.ics, 6, 373-382; U.S. Pat. Nos. 6,787,308; and 6,833 ,246; herein incorporated by reference in their entireties), the Lynx massively parallel signature sequencing technology (Brenner ei al. (2000), Nat. Biotechnol. 18:630-634; U.S. Pat. Nos. 5,695,934; 5,714,330; herein incorporated by reference in their entireties), and the Adessi PCR colony technology (Adessi el al. (2000). Nucleic Acid Res. 28, E87; WO 2000 / 018957; herein incorporated, by reference in its entirety).| '0070] Also provided are reaction mixtures formed according to the methods described herein. For example exemplary reaction mixtures can comprise the hydrogel beads linked to cell binding agents as described herein, optionally mixed with a population of cells. In some embodiments; the number of hydrogel beads in the reaction mixture is higher than the number of cells. For example, the number of hydrogel beads can be at least 2, 4, 6, 8, 10 (e.g., 2-20) times the number of cells in the reaction mixture. This can be helpful for example in improving the portion of cells bound by at least one bead. In some embodiments, the reaction mixture is an aqueous solution. In some embodiments, the reaction mixture is in partitions (e.g., droplets or microwells).|007t| The kit can include any of the components described herein with regard to the methods described herein. For example, kits can comprise a plurality of hydrogel beads linked to cell binding agents as described herein. Optionally the kits can comprise other reagents in the same or different container than contains the hydrogel beads. In some embodiments, a second container containing a cell lysis buffer is provided. In some embodiments, the cell-lysis buffer comprises a detergent, for example as described elsewhere herein.|0072| The kits can further comprise one or more restriction enzymes, endonucleases, exonucleases, ligases, polymerases, RNA polymerases. DN A polymerases, reverse transcriptases, topoisomerases, kinases, phosphatases, buffers, salts, metal ions, reducing23TO WE SEN D 7S7&IS84 1agents, BSA, spermine, spermidine, glycerol, oligonucleotides, primers, probes, or labels(e.g., fluorescent labels). The kits can comprise one or more sets of instructions.EXAMPLESExample 1|0073| This example shows hydrogel beads linked to cholesterol were determined to bind to cells before and into compartmentalization in an emulsion in oil. The hydrogel beads we composed of polyacrylamide chains crosslinked with bis-acrylamide. DNA oligonucleotides containing acrylic groups covalently linked to rhe 5-prime end of the DNA molecule were polymerized into the polyacrylamide chains during polymerization of the hydrogel beads. The DNA oligonucleotide was then ligated to another DNA oligonucleotide containing a triethylene glycol linker between the 3-prime end of the DNA oligonucleotide and the cholesterol molecule. Beads, with and without (control) cholesterol Linked to the beads, and cells were, paired by pipet mixing and incubating at room temperature for 5 minutes in a standard PBS solution to pair them. For experiments shown, beads were added at a tenfold higher quantity then cells to ensure beads bound to cells. A standard RT reaction without detergent was mixed Into the solution before compartmentalization by formation of an emulsion having aqueous droplets in oil. The protocol was substantially as found in Hatori, er cd , dttd C / iem. 2018, 90, 16, 98'13-9820. Briefly, the PBS solution containing cells and beads were mixed with a 2x RT reaction mixture on ice. Hydrofluoroether, such, as HFE« 7100, was added to the cell, bead, and RT mixture with a 10-fold excess volume. Next, the sample was vortexed on a standard lab vortex to create an emulsion. The emulsion was incubated for 1 hour at 37 C. Next, the emulsion was imaged on a microscope and the cells and bead counted to determine if the droplet compartment contained a cell and a bead. Samples were examined to determine the percentage of cells in droplets with beads. In droplets with beads containing no cholesterol 40 % of the cells were in droplets with beads. However, in droplets with beads containing cholesterol linked to the 3-prime end of an oligonucleotide by two triethylene glycol spacers (i.e,, a hexaethylene glycol spacer), or three triethylene glycol (i.e., a nonethylene glycol spacer) spacers droplets with cells and beads increased significantly. Quantification of the beads containing the proper oligo nucleotide containing cholesterol was determined by ddPCR with about 5- 10 million cholesterol containing oligos per bead for these experiment (data not shown). See, FIG. 3.Example 224TOWXSEND 7S70IS84 1[0074| This example shows that, hydrogel beads linked to cholesterol and carrying oligonucleotides bind to cells and can capture and be used for synthesis of GAPDH cDNAs from the cells. Cells and beads containing cholesterol via a hexaethylene glycol spacer to an oligonucleotide were paired into droplets as described in Example 1 , Optionally beads containing additional triethylene glycol spacers between the oligonucleotide and the cholesterol molecule were incubated with cells in a standard PBS solution as described inExample 1. A standard RT reaction with detergent was mixed into the solution before compartmentalization. Next the emulsion was incubated for 1 hour at 37* C to promote cellular cDN'A synthesis. After the cDNA synthesis step, the emulsion was broken and GAPDH cDN A was quantified by ddPCR. The cholesterol-linked beads produced more GAPDH copies in both the Igepal and Tween20 detergents. In the samples containing NP40 and TritonXIOO the amount of GAPDH detected were similar to controls. See. FIG. 4.[00751 It is understood that the examples ;uid embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this appl ication and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.25TOWXSEND 7S7&I584 1

Claims

WHAT IS CLAIMED IS:1 . A method of forming partitions containing a hydrogel bead and a cell or forming a cross-linked hydrogel surrounding physically-separated cell / hydrogel bead pairs, the method, comprising, providing a mixture of cells and hydrogel beads, wherein the hydrogel beads are linked to a plurality of oligonucleotides, wherein a cell-binding agent is linked to the hydrogel beads via a linker comprising a non-nucleic acid linker or wherein the ce ll-binding agent is linked to an oligonucleotide that anneals to one of the plurality of oligonucleotides linked to the hydrogel bead, wherein the cell-binding agent binds to a cell to form cell / hydrogel bead pairs such that some cells are linked to the hydrogel beads via the cellbinding agent; and introducing the cell / hydrogel bead pairs into partitions or separating the cell / hydrogel bead pairs in a cross-linked hydrogel surrounding the cell / hydrogel bead pairs, thereby forming partitions containing a hydrogel bead and a cell or a cross-linked hydrogel surrounding physically-separated cell / hydrogel bead pairs.

2. The method of claim 1 , wherein the linker further comprises a. nucleic acid,3. The method of claim 1 or 2, wherein the non*nucleic acid linker comprises ethylene glycol, prqpanediol-S-succinic acid, polyethylene glycol, abasic furan or a tetrahydroluaran derivative mimicking an abasic site, or a polyandioL4. The method of claim 2, wherein the ethylene glycol is triethylene glycol, hexaethylene glycol, or nonethylene glycol,5. The method of any one of claims 1 -5, wherein the non-nucleic acid linker is at least 2, 4, 6, 8, .10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36, (e.g., 2-36) carbons in length,6. The method of claim 1, further comprising, after the providing and before the introducing, removing from the mixture (a.) at least some hydrogels beads that do not bind to the cells, (b) at least some cells that do not bind to the hydrogels beads, or (c) both (a) and (b).26TOWISSEND 7S76HS4 iThe method of claim 1 , wherein the cell-binding agent is a cholesterol moiety, a lipid moiety, a cell-adhesion polypeptide, a lectin or an antibody that binds to the cell,8. The method of claim 7, wherein the antibody is biotinylated and the hydrogel bead is linked to avidin or streptavidin,9. The method of claim 1 or 7, wherein the cell-binding agent is covalently-linked to the hydrogel bead.I0. The method of any one of claims 1 -9, wherein the cell-binding agent is linked to one or more oligonucleotide from the plurality of oligonucleotides, wherein the one or more oligonucleotide is linked to the cell-binding agent via the non-nucleic acid linker,I I . The method of claim 1 , wherein the cell-binding agent is linked to an oligonucleotide that anneals to one of the plurality of oligonucleotides linked to the hydrogel bead,12. The method of any one of claims 1-1 1 11 , wherein at least a majority of the plurality of oligonucleotides linked io the hydrogel beads comprise a barcode sequence unique for the bead to which the oligonucleotides are linked.

13. The method of any one of claims 1-12, further comprising lysing the cell in the partitions.

14. The method of claim 1.3, wherein the cells are lysed with a detergent,15. The method of claim 14, wherein the detergent is present in or introduced into the partition.

16. The method of claim 14 or 15, wherein the detergent is selected from the group consisting of Tween-20, Triton-X and NP40,17. The method of any of one of claims 1-16, wherein the hydrogel beads comprise a magnetic or paramagnetic particle.

18. The method of any one of claims 1 - 16, wherein the partitions are microwells or aqueous droplets in an emulsion.27TOWNSEND 7S76I5S4 I19. A hydrogel bead linked to a plurality of oligonucleotides, wherein a cell-binding agent is linked to the hydrogel beads via a linker comprising a non-nucleic acid linker or wherein the cell-binding agent is linked to an oligonucleotide that anneals to one of the plurality of oligonucleotides linked to the hydrogel bead. 20, The hydrogel bead of claim 19, wherein the linker further comprises a nucleic acid, 21. The hydrogel bead of claim 19 or 20, wherein the non-.nncleic acid linker comprises ethylene glycol, propanediol-3-succmie acid, polyethylene glycol, abasic furan or a tehahydrofuaran derivative mimicking an abasic site, or a polyaudioL 22. The hydrogel bead of claim 21 wherein the ethylene glycol is triethylene glycol or hexaethylene glycol or nonethylene glycol.

23. The hydrogel bead of any one of claims 19-22, wherein the non- nucleic acid linker is at least 2, 4. 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36, (e.g., 2-36) carbons in length.

24. The hydrogel bead of claim 19, wherein the cell-binding agent is a cholesterol moiety, a lipid moiety, a cell-adhesion polypeptide, a lectin or an antibody that binds to the cell.

25. The hydrogel bead of claim 24, wherein the antibody is biotinylated and the hydrogel bead is linked to avidin or streptavidin.

26. The hydrogel bead of claim 19 or 24, wherein the cell-binding agent is covalently-linked to the hydrogel bead.

27. The hydrogel bead of any one of claims 19-26, wherein the cell- binding agent is linked to one or more oligonucleotide from the plurality of oligonucleotides, 28. The hydrogel bead of claim 19, wherein the cell-binding agent is linked to an oligonucleotide that anneals to one of the plurality of oligonucleotides linked to the hydrogel bead.28TOWNSEND 7S7SHS4 129. The hydrogel bead of any one of claims 19-28, wherein at least a majority of the plurality of oligonucleotides linked to the hydrogel beads com prise a barcode sequence unique for the bead to which the oligonucleotides are linked.30, A reaction mixture comprising a cell and the hydrogel bead of any one of claims 19-29.31 . A kit comprising one or more hydrogel bead of any one of claims 19-29.

32. The kit of claim 31 , further comprising a detergent.

33. The kit of claim 32, wherein the detergent is selected from the group consisting of wherein the detergent is selected from the group consisting of Tween-20, Triton-X and NP40.

34. A surface comprising discrete locations linked via a linker comprising a non-nucleic acid linker to a cell-binding agent or wherein the cell-binding agent is linked to an oligonucleotide that anneals to an oligonucleotide linked to the surface.

35. The surface of claim 34, wherein the linker further comprises a nucleic acid.

36. The surface of claim 34 or 35, wherein the non-nucleic acid linker comprises ethylene glycol, propanediol-3 -succinic acid, polyethylene glycol abasic furan or a tetrahydrofuaran derivative mimicking an abasic site, or a polyandiol.The surface of claim 36, wherein the ethylene glycol is triethylene glycol, hexaethylene glycol or nonethylene glycol.

38. The surface of any one of claims 34-37, wherein the non-nucleic acid linker is at least 2, 4, 6, 8, 10, 12. 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36, (e.g., 2-36) carbons in length.

39. The surface of claim 34, further comprising cells linked to cel l-binding agents.29TOWNSEND 7S76ISS4 i40. The surface of claim 39. further comprising a layer of cross-linked hydrogel surrounding the cells.

41. The surface of any one of claim 34-40, wherein the cell-binding agent is a cholesterol moiety, a lipid moiety, a cell-adhesion polypeptide, a lectin or an antibody that binds to the cell.

42. The surface of any one of claims 34-41 , wherein the oligonucleotides comprise a barcode sequence unique for the discrete location to which the oligonucleotides are linked.30TO WE SEND 7S7&IS84 1

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