Method for capturing cellular analytes

Abstract

The invention relates to methods for capturing cellular analytes, particularly for the generation of libraries and for single cell sequencing. The methods comprise using temperature control to convert molten hydrogel mixed with cellular analytes and cellular analyte capture agents to hydrogel beads in which captured cellular analytes are embedded. The invention also relates to methods of single cell sequencing or library generation, and to hydrogel beads, compositions, kits, devices, and chips for use in the methods.

Description

[0001] METHOD FOR CAPTURING CELLULAR ANALYTES

[0002] Field of the invention

[0003] The invention relates to methods of capturing cellular analytes, particularly from single cells, for library generation and sequencing. The invention also relates to methods of single cell sequencing or library generation and to hydrogel beads, compositions, kits, devices, and chips for use in the methods of capturing cellular analytes, single cell sequencing and library generation.

[0004] Background to the invention

[0005] Single cell library preparation methods typically involve isolating cells in aqueous droplets in oil (a water in oil emulsion) using a microfluidics system, lysing the cells in the droplets, and amplifying and sequencing analytes from the cell. Barcode sequences are used to identify individual amplicons or the analytes from a particular cell. There are variations of the method suited to different purposes, but common limitations include high cost, low throughput, low efficiency, high background to noise ratios, high sample loss, high error rates, low fidelity, and high complexity to the methods.

[0006] These limitations may be further compounded by the particular requirements of specific experimental objectives. For example, understanding immunity to infectious pathogens, cancer antigens, autoimmune targets and multiple other immune responses requires the detailed molecular characterisation of the human receptors that recognise the targets. For antibody responses this is the ‘B cell receptor’ (BCR) and for T cell responses this is the ‘T cell receptor’ (TCR). This information is highly relevant to new drug discovery, vaccines and immunotherapies. Full analysis of BCRs and TCRs requires knowing the sequences of the two receptor chains (alpha and beta for TCRs, light and heavy for BCRs) and how these pair together. Until recently, technologies were not able to deliver this. There has been significant growth in technologies now available to deliver paired sequencing of TCRs and BCRs, some of which are commercially available. However, current assays are limited by cost, limitations of total cell numbers analysed, and ability to pair the chains. Current high-throughput (up to and above a million individual cells) technologies usually result in the loss of transcript pairing, while methods which preserve pairing often have extremely high costs and low throughput (5-10,000 cells), see e.g. WO2019 / 023627. Hence, there remains a need for cost-effective, high-throughput methods for single cell library preparation, e.g. for paired sequencing of BCRs and TCRs and other similar methods.

[0007] Summary of the invention

[0008] The present inventors have developed a new high throughput, high fidelity and cost- efficient method of capturing cellular analytes. The method is particularly useful for single cell library preparation and sequencing applications and for identifying paired analytes in single cells. The method uses a hydrogel, such as agarose, and temperature controls to melt and re-soli dify the hydrogel during the method. Changes in temperature are used to control the capture and release of agents and analytes that are combined with the hydrogel. When molten, the hydrogel can be mixed with other reagents and analytes. When cooled, the hydrogel forms a porous (solid) gel in which other components of the aqueous mixture are captured. Microfluidics may be used to form an emulsion of aqueous droplets, including molten hydrogel, in oil. When cooled, the hydrogel crosslinks within the droplet, forming a porous gel bead in which other components of the aqueous mixture are captured. This allows the contents of the original droplet to be processed outside of the emulsion without being separated and dispersed. It further allows the original drop contents to be re-encapsulated in a new emulsion droplet and for new components, such as barcode nucleotides, to be added at this stage.

[0009] For example, the method can be used to create an RNA-capture hydrogel bead comprising embedded poly(dT) beads. The RNA-capture hydrogel beads have a novel design which uniquely does not require the conjugation of oligos to agarose, but rather the passive capture of RNA through smaller poly(dT) beads trapped inside a hydrogel matrix. The use of RNA-capture hydrogel beads (or other capture agents) ensures that the product of interest is captured in the bead, while allowing the remainder of the cell lysate to diffuse out of the bead. This is advantageous as it prevents the accumulation of cell lysate components which may be inhibitory to downstream PCR and sequencing steps. This method is also cost-effective, ensures high-fidelity and allows faster processing compared to existing methods. For single cell sequencing application, the method can result in all mRNA from one cell being localised in one bead, allowing cellular barcoding to make the assay truly quantitative.

[0010] The present inventors have also combined the new method of cellular analyte capturing with a microfluidics-based approach to single cell sequencing which allows fusion of two independent gene transcripts within single droplets (maintaining single cell resolution) and introduces barcodes to underpin the single cell resolution. The method allowed sequencing of paired analytes, such as RNA transcripts (for example B cell and T cell receptors) at very high throughout (105to 106cells), single cell resolution, high fidelity and low cost. Previously described high-throughput single-cell TCR / BCR sequencing methods (e.g. the method described in WO2019 / 023627) do not enable this barcoding.

[0011] Accordingly, in a first aspect, the invention provides a method of capturing cellular analytes, the method comprising the steps of:

[0012] (a) forming an emulsion by dispersing a first aqueous fluid phase comprising a suspension of cells and a second aqueous fluid phase comprising lysis buffer, a molten hydrogel and cellular analyte capture agents into a flowing oil phase simultaneously, such that the aqueous phases form droplets within the flowing oil; and

[0013] (b) cooling the droplets such that hydrogel beads are formed within the droplets, the hydrogel beads being porous and comprising cellular analytes from the cell lysate captured by the cellular analyte capture agents within the pores of the hydrogel beads, wherein the cellular analyte capture agents are not covalently attached to the hydrogel (bead).

[0014] In a second aspect, the invention provides a method of single cell sequencing, comprising capturing cellular analytes in a hydrogel bead according to a method of the invention.

[0015] In a third aspect, the invention provides a hydrogel bead comprising at least one poly(dT) bead. The poly (dT) bead is not covalently attached to the hydrogel bead.

[0016] In a fourth aspect, the invention provides the use of a hydrogel bead according to the invention.

[0017] In a fifth aspect, the invention provides a composition or kit comprising a hydrogel, poly(dT) beads and lysis buffer. The poly (dT) bead is not covalently attached to the hydrogel bead.

[0018] In a sixth aspect, the present invention provides a device configured to perform a method of the invention.

[0019] In a seventh aspect, the present invention provides a chip configured for carrying out a method of the invention.

[0020] The invention will now be described in more detail, by way of example and not limitation, and by reference to the accompanying drawings. Many equivalent modifications and variations will be apparent, to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the disclosure set forth are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the scope of the invention which is defined by the claims. All documents cited herein, whether supra or infra, are expressly incorporated by reference in their entirety. The present disclosure includes the combination of the aspects and features described except where such a combination is clearly impermissible or is stated to be expressly avoided. Section headings are used herein for convenience only and are not to be construed as limiting in any way.

[0021] Description of the Figures

[0022] Figure 1: Overview of technology

[0023] Figure 1 shows a microfluidics cellular analyte capture method of the invention. Two aqueous phases, the first comprising a suspension of cells and the second comprising lysis buffer, molten hydrogel and cellular analyte capture agents are injected simultaneously into a flowing oil phase. This results in the formation of droplets comprising cell lysate, hydrogel and cellular analyte capture agents. The droplets are cooled such that hydrogel beads are formed within the droplets. The beads are washed to remove cell lysate before reencapsulation into droplets with PCR mix comprising barcode sequences.

[0024] Figure 2: Barcoding strategy

[0025] Figure 2 shows a barcoding strategy according to the invention. Hydrogel beads comprising cellular analyte capture agents and cellular analytes are mixed with PCR mix and barcode sequences, before being injected into a flowing oil phase to form droplets. Reverse transcription and overlap extension PCR are then performed in-drop in order to link two transcripts of interest, as well as the barcode sequence, into a single DNA or cDNA molecule. Figure 3: TCR single cell sequencing method

[0026] Figure 3 shows a TCR single cell sequencing method according to the invention. Using the microfluidics cellular analyte capture method described in Figure 1, mRNA from a T cell sample are captured within the hydrogel bead of the invention by means of the poly(dT) beads within it. Each droplet comprises the cell lysate from a single T cell. In-drop overlap extension PCR is used to link the alpha and beta chains and introduce a barcode sequence. The droplets are then pooled for nested PCR and library preparation followed by sequencing. Figure 4: Imaging of beads and RNA capture demonstration

[0027] Figure 4 shows hydrogel beads of the invention with poly(dT) beads trapped within them. Brightfield microscopy shows poly(dT) beads trapped within the agarose bead. Fluorescence microscopy using a poly-A fluorescent probe (FAM) shows successful poly -A RNA capture.

[0028] Figure 5: Chip design

[0029] Figure 5 shows the design of the microfluidics chips utilised in the invention.

[0030] Figure 6: Experimental data demonstrating fidelity and sensitivity

[0031] Proof of concept mixes to test for fidelity and sensitivity. Fidelity and doublet error rate were ascertained via mixes of cell lines with known BCR / TCR sequences. Error was calculated as the proportion of cell line chains paired with non-native sequences. A) 50-50 mix between cell line 355-3C (expected IGHV4-34, IGLV1-51 gene usage) and 355-9C (IGHV1-69, IGKV3). B) 90-10 mix between 355-3C and 355-9C C) Jurkat cell line spiked-in at 1% to T cells from a healthy donor. D) 50% HBP-ALL cell line spike-in with T cells from donor. E) 10% spike-in of HBP-ALL cell line to T cells from healthy donor. Error rate of 0.18%- 10%.

[0032] Figure 7: Fidelity comparison with Dekosky method

[0033] A) Figure 7A shows the fidelity of the Dekosky single cell paired sequencing method, where fidelity is the percentage of correctly paired alpha and beta J gene pairs. Dekosky data was obtained from Fahad, A.S., Chung, C.Y., Lopez Acevedo, S.N. et al. Cell activation-based screening of natively paired human T cell receptor repertoires. Sci Rep 13, 8011 (2023). https: / / doi.org / 10.1038 / s41598-023-31858-4. B) Figure 7B shows the fidelity of the method of the invention utilising agarose beads. C) Figure 7C shows the fidelity scores of four separate experiments on T cells from healthy donors and their resulting fidelity distributions.

[0034] Description of the Sequences

[0035] SEQ ID NOs 1 to 78 set forth the heavy and light chain CDR3 sequences shown in Table 1.

[0036] Detailed Description

[0037] Cellular analytes

[0038] The present invention relates to a method of capturing cellular analytes. In some embodiments, the cellular analytes may be selected from polynucleotides such as DNA, cDNA and / or RNA, or from polynucleotides, oligonucleotides, DNA, RNA, mRNA, proteins, polypeptides and / or peptides, or cell surface receptors. Most typically, the cellular analytes are mRNA.

[0039] The cellular analytes may be derived from any cell, for example a bacterial cell, plant cell, or animal cell, such as a mouse cell, rabbit cell, camel cell, or most typically, a human cell. In some embodiments, the cellular analytes are derived from an immune cell, such as a T cell or a B cell. In some embodiments, the cellular analytes are derived from a cancer cell.

[0040] The cellular analytes may be derived from any suitable sample. Specific examples include a sample of cells, cell nuclei or cellular vesicles, a single cell, a single cell nucleus, a single vesicle, a tissue sample or tissue section, or a biological fluid sample, optionally blood, a blood fraction, serum, plasma, saliva or urine sample.

[0041] Cellular analytes and capture agents

[0042] The present invention makes use of cellular analyte capture agents. A cellular analyte capture agent is an agent that is able to bind to a cellular analyte during the method of the invention in order to isolate them for further analysis. For example, a cellular analyte capture agent may remain bound to a cellular analyte during a step of washing. The cellular analyte capture agent may play a role in retaining the cellular analyte of interest within the hydrogel bead, for example during a step of washing, such that the cellular analyte remains associated with the hydrogel bead and / or droplet in subsequent steps.

[0043] A cellular analyte capture agent may typically comprise a microbead and an array of oligonucleotides. The oligonucleotides, or an analyte capture region thereof, are typically able to bind to the cellular analytes of interest by nucleotide base pairing. The oligonucleotides / capture region are typically at least 10, or at least 15, 20 or 25 nucleotides in length, such as from about 15 to about 50, from about 20 to about 40 or from about 25 to about 35 nucleotides in length. In some cases, the oligonucleotides may comprise one or more nucleotide analogues, as are known in the art, that form double-stranded hybrids with higher stability than natural nucleotides. In this case, the oligonucleotides could be shorter, for example between 3 and 50, or 40 or 30 or 25 nucleotides in length. The oligonucleotide is capable of hybridizing to target analyte such that analyte sequence can be amplified as described herein.

[0044] In some embodiments, the analyte capture region is a polythymidine. Polythymidine may hybridise to and capture any polynucleotide in the sample that comprises a suitable polyadenosine, such as polyadenylated mRNA. Typically, the polythymidine may be at least 10, or at least 15, 20 or 25 or 30 thymidines in length, such as from about 15 to about 50, from about 20 to about 40 or from about 25 to about 35 thymidines.

[0045] In some cases, the analyte capture region(s) may comprise or consist of a nucleotide sequence designed to hybridise to a complementary sequence in a target polynucleotide analyte.

[0046] In another exemplary embodiment, the oligonucleotide is for capturing / hybridising to transposed DNA. In this case an analyte capture region may comprise or consist of a sequence that is complementary to transposed DNA in a sample, for example to a transposed MEDS DNA sequence. In some cases, the sequence may be gene or transcript-specific, such as a polynucleotide sequence that is complementary to, or at least 80%, 85%, 90%, 95%, 98% or 99% complementary to, a viral sequence, a bacterial sequence or a sequence associated with a disease or disorder, such as a sequence from a cancer-associated antigen or a neoantigen.

[0047] In another exemplary embodiment, the oligonucleotide may comprise or consist of a biotinylated nucleotide sequence. Nucleotides or polynucleotides may be biotinylated using methods known in the art. Typically, the biotinylated sequence may be at least 10, or at least 15, 20, 25 or 30 nucleotides in length, such as from about 15 to about 50, from about 20 to about 40 or from about 25 to about 35 nucleotides. A biotinylated capture region may be used to capture any suitable target analyte comprising streptavidin or avidin.

[0048] In another exemplary embodiment, the oligonucleotide may comprise or consist of an aptamer. Aptamers can be produced using SELEX (Stoltenburg, R. et al., (2007), Biomolecular Engineering 24, p381-403; Tuerk, C. et al., Science 249, p505-510; Bock, L. C. et al., (1992), Nature 355, p564-566) or NON-SELEX (Berezovski, M. et al. (2006), Journal of the American Chemical Society 128, pl410-1411). Typically, an aptamer may be at least 15 nucleotides in length, such as from about 15 to about 50, from about 20 to about 40 or from about 25 to about 30 or nucleotides in length. An aptamer may bind to analyte such as small molecules, proteins, nucleic acids or cells. Aptamers may be designed or selected to bind to pre-determined target analyte(s).

[0049] In another exemplary embodiment, the oligonucleotide may be designed to capture a polynucleotide tag added to analyte of interest. Typically, a micro-bead is approximately spherical or sphere-like. Microbeads are typically less than 500 pm, or less than 400 pm, 300 pm or 200 pm in diameter, for example, between about 1 and 500 pm, 1 and 200 pm, 5 and 100 pm, 5 and 50 pm, or 10 and 40 pm. Microbeads with surface-conjugated polynucleotides, as described below, are typically about 1 to 50 pm, or more typically about 1 to 15 pm or 8 to 20 pm in diameter. Such micro-beads are well-known in the art and may be made from, for example, a biocompatible polymer such as polystyrene, polyacrylamide or hydroxylated methacrylic polymer, or from controlled pore glass.

[0050] According to the present invention, the capture agents are trapped in porous hydrogel beads as they form. In particular, the capture agents are trapped in the hydrogel bead pores because of the size and / or shape of the capture agents relative to the size and / or shape of the hydrogel bead pores. This is advantageous as it means that the cellular analyte capture agents, and any cellular analytes captured by them, remain within the hydrogel beads during any wash steps, while unbound cellular analytes and other reagents flow out of the beads. In contrast, known methods for capturing cellular analytes typically make use of smaller capture agents, such as nucleotides. Due to their small size, such nucleotide capture agents would not be retained in the pores of the hydrogel and therefore are typically conjugated to the agarose such that they are retained during any wash steps. Advantageously, the methods of the invention do not require conjugation of any nucleotides to hydrogel, e.g. agarose.

[0051] Hence, the cellular analyte capture agents have a size and / or shape that allows them to be (physically) trapped in the pores of a hydrogel as it solidifies. In the methods of the invention, the size and / or shape of the cellular analyte capture agents is such that they are trapped in the pores of the hydrogel as it solidifies. For example, capture agents having a diameter of at least about 0.5 pm or about 1 pm may typically be suitable, e.g. when using agarose hydrogel, or other hydrogels that form beads having similar-sized pores. For example, capture agents, or more specifically, capture agent beads as described below, having a diameter of about 0.5-5 pm, or more typically about 1-2 pm in diameter (such as the poly(dT) beads described below) may be used. In some embodiments, the capture agents / beads may have a diameter of about 0.5-20 pm, 0.5-10 pm, 0.5-5 pm, or 1-20 pm, 1- 10 pm or 1-5 pm.

[0052] Microbeads with surface-conjugated polynucleotides for cellular analyte capture are suitable for use with some embodiments of the invention. In some embodiments, the oligonucleotides may include identifier / barcode sequences. However, these relatively larger micro-beads (typically between 50 and 500 pM in diameter and comprising between 105and 1012polynucleotides) with surface-conjugated (optionally barcoded) polynucleotides, which may in some cases be handled and processed outside of an emulsion after analyte capture, are in general time-consuming and expensive to manufacture. Hence, a key advantage of some embodiments of the invention is that these more complex cellular analyte capture agents can be replaced by smaller and simpler polynucleotide-containing capture agents, such as the poly(dT) beads described below. Instead, the whole solidified hydrogel bead may be used to gather multiple capture agents (e.g. poly(dT) beads) such that they are isolated and processed together, whilst keeping the analytes from a particular sample (e.g. a single cell) united.

[0053] Hence, in some embodiments, the cellular analyte capture agents are poly(dT) beads, e.g. oligo(dT)25, which are low-cost and readily available. The poly(dT) beads may be magnetic or paramagnetic to aid with handling. The beads are covalently attached to oligonucleotides comprising polythymidines. The beads are typically smaller than those described above with surface-conjugated oligonucleotides, typically about 0.5-5 pm, or more typically 1-2 pm in diameter. These polythymidines are able to hybridise with the polyA tails of cellular mRNA molecules, thereby capturing them. Handling hydrogel beads having internal capture elements also has the advantage of reducing the likelihood of RNA contamination and assay noise compared to handling isolated microbeads with surface- conjugated oligonucleotides and captured cellular analytes.

[0054] Method of capturing cellular analytes

[0055] In the method of the invention, molten hydrogel may be mixed with cellular analyte capture agents, a suspension of cells (or other source of cellular analytes as described herein), lysis buffer and oil such that the mixed aqueous phase forms droplets in an oil emulsion.

[0056] More specifically, an emulsion may be formed by dispersing an aqueous fluid phase comprising a suspension of cells or the like, with an oil phase and one or more further aqueous phases comprising molten hydrogel, lysis buffer and cellular analyte capture agents. Typically, separate streams of the aqueous phases are combined into a flowing oil, such that the combined aqueous phases form droplets within the flowing oil. This is typically achieved using a microfluidics approach or a microfluidics device. Suitable microfluidics approaches which may be used or adapted for use in the present invention are described in, for example, WO2019 / 023627.

[0057] When a suspension of cells, or similar membrane-bound cellular components (e.g. cell nuclei or vesicles) is mixed with lysis buffer, the cells / nuclei / vesicles are lysed and analytes are released into the aqueous phase. Here they come in contact with and bind to the cellular analyte capture agents.

[0058] The emulsion is formed at a temperature that maintains the hydrogel in a molten state. The droplets are then cooled to a temperature at which the hydrogel polymers cross-link to form a porous (solid) gel bead. Typically, part of the water in the droplet is bound to the polymer (by hydrogen bonds), part is in an intermediate state, and part is free (not bound) but nonetheless trapped within the pores of the hydrogel bead. Hence, when the emulsion is cooled and the hydrogel beads are formed, captured analytes are trapped within the hydrogel bead.

[0059] An unexpected advantage of the present invention is that the creation of the hydrogel beads comprising cellular analyte capture agents allows for an increase in the throughput and fidelity of the method compared with prior art methods (e.g. WO2019 / 023627).

[0060] For single cell sequencing applications, it is beneficial to maximise the number of droplets in the emulsion that contain a single cell, and minimise the number of droplets that contain either more than one cell, or no cell. This assists with single cell resolution, for example when sequencing the captured cell analytes, and can be achieved by, for example, modifying the concentration of the cell suspension, the flow rate(s), and / or the geometry of the microfluidics chip . In some embodiments, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% of the droplets, or of the droplets that encapsulate any one or more cells, comprise cell lysate from a single cell.

[0061] Additionally, as all of the capture elements within a single drop are captured inside a single hydrogel bead, this allows for use of many capture agents per drop without a corresponding loss of fidelity. For example, previous methods which aim to provide a single capture agent per droplet, e.g. a single microbead with covalently attached polynucleotides, would necessarily result in droplets comprising more than one microbead and droplets with no microbead. Droplets with multiple microbeads would lead to an artificial increase in signal for particularly sequences, while RNA from cells in a droplet comprising no microbead would not be captured for analysis. In some embodiments, the present invention provides a method of capturing cellular analytes, the method comprising the steps of:

[0062] (a) forming an emulsion by dispersing a first aqueous fluid phase comprising a suspension of cells and a second aqueous fluid phase comprising lysis buffer, a molten hydrogel and cellular analyte capture agents into a flowing oil phase simultaneously, such that the aqueous phases form droplets within the flowing oil; and

[0063] (b) cooling the droplets such that hydrogel beads are formed within the droplets, the hydrogel beads being porous and comprising cellular analytes from the cell lysate captured by the cellular analyte capture agents within the pores of the hydrogel beads.

[0064] Hydrogel

[0065] Hydrogels are crosslinked polymer chains with three-dimensional (3D) network structures, which can absorb relatively large amounts of fluid. Biocompatible hydrogels are capable of trapping biological molecules within their matrix without causing them to degrade or denature. The skilled person will understand that any suitable biocompatible hydrogel can be used in the method of the invention. In some embodiments, the hydrogel is selected from the group consisting of PEGA, poly-acrylamide, alginate, agarose, and collagen. In some embodiments, the hydrogel is agarose. An ultra-low gelling temperature agarose may be used, such as 3,6-Anhydro-a-L-galacto-P-D-galactan (e.g. Sigma #A5030 Ultra-low Gelling Temperature Agarose).

[0066] The hydrogel can be selected to have suitable melting and solidifying temperatures. Melting and solidifying should occur within a range that can be applied to the experimental apparatus being used and that does not interfere with the integrity of the sample / analytes, the functioning of the equipment (e.g. flow through a microfluidics device) or with the processing or amplification steps. It is typically convenient if the hydrogel is solid at room temperature. However, in some embodiments, it may be convenient if the hydrogel is liquid at room temperature. Typically, the melting temperature is below the temperatures used for a PCR reaction used in an amplification step. Typically, the hydrogel will have a melting temperature of between about 40°C and 60°C. Typically, the hydrogel will have a solidifying temperature of between about 0°C and 40°C, more typically between 0°C and 20°C. An exemplary hydrogel is Ultra-low Gelling Temperature Agarose, such as Sigma #A5030. However, the skilled person can readily identify alternative hydrogels that could be used. Heating and Cooling

[0067] In the method of the invention, the emulsion is formed at a temperature that maintains the hydrogel in a molten state, and then cooled such that hydrogel beads are formed within the droplets. The skilled person will understand that the temperature at which these are carried out will depend on the hydrogel used and it is within the capabilities of the skilled person to determine the temperatures that should be used. For example, when using agarose Sigma #A5030 Ultra-low Gelling Temperature Agarose as the hydrogel, the emulsion may in exemplary embodiments be formed at a temperature of between 45°C and 60°C, and the cooling step may be carried out at a temperature of between about 0 and 10 °C, about 2 and 6 °C, or about 3 and 5 °C. In some embodiments, the step of cooling comprises cooling at around 4 °C.

[0068] Washing and re-encapsulating

[0069] In some embodiments, the method further comprises breaking the droplets. Breaking of the droplets may comprise, for example, administration of perfluoro- 1 -octanol, for example lH,lH,2H,2H-Perfluoro-l-octanol. The hydrogel beads may be combined and processed in bulk, without separating the majority of the analytes associated with the original liquid droplet, i.e. all of those trapped within the hydrogel bead. Where a droplet includes a single cell, the majority of the analytes of the cell remain within the same hydrogel bead. The beads may be washed to remove oil and any excess reagents and cellular components that are not trapped within the hydrogel beads, which might otherwise inhibit downstream PCR and sequencing steps. The beads may be washed using, for example, a buffer, for example lysis buffer. The beads may then be re-suspended in an aqueous composition.

[0070] In some embodiments, the hydrogel beads are re-encapsulated in a second oil emulsion. A microfluidics approach may be used. To prevent mixing of the contents of different beads, the emulsion breaking, washing / other processing steps and re-encapsulation are carried out at a temperature below the melting temperature of the hydrogel beads. As with the first encapsulation, in some embodiments it is desirable to maximise the encapsulation of single hydrogel beads in each droplet, and to minimise the number of droplets that contain multiple hydrogel beads or no hydrogel beads. In some embodiments, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% of the re-encapsulated droplets, or of the re-encapsulated droplets that contain any hydrogel beads, comprise a single hydrogel bead only.

[0071] When re-encapsulating the hydrogel beads, it is possible to include additional components (for example, PCR reagents, and / or barcoding elements) that are either mixed with the suspension of hydrogel beads before forming the emulsion, or that are included in a second aqueous phase dispersed with the suspension of hydrogel beads as the emulsion is formed. This is a particular advantage of the invention. The use of the hydrogel allows the analytes to be captured in a first step involving essentially simultaneous lysis of the cells and analyte capture and formation of a first emulsion, and for the contents of each droplet formed in this first step to remain together during formation of the second emulsion and the addition of separate components. Each of the two emulsion forming steps can be controlled separately. This is particularly useful, for example, for adding barcoding elements at a limiting dilution to the re-encapsulated hydrogel beads during the second emulsion formation. This is described further below.

[0072] Method of cellular analyte library preparation and single cell sequencing

[0073] In some embodiments, the present invention provides a method of cellular analyte library preparation and / or single cell sequencing. The method comprises capturing cellular analytes in a hydrogel bead according to the method of the invention as described herein. Single cell sequencing refers to a method of analysing the cellular analytes at the level of individual cells. For example, single cell sequencing can be used to measure the number of cells expressing a given BCR or TCR. This may be achieved by incorporating a unique barcode into the cellular analytes of each cell, such that the origin of the analytes can be identified during downstream analysis.

[0074] In some embodiments, the method of library generation and / or single cell sequencing comprises the steps of:

[0075] (a) capturing cellular analytes on cellular analyte capture agents within hydrogel beads using a method according to the invention, optionally wherein the cellular analytes are DNA or RNA;

[0076] (b) breaking the droplets and washing the hydrogel beads to remove excess cell lysate not captured within the agarose beads;

[0077] (c) mixing the hydrogel beads with PCR reagents and primers; (d) melting the hydrogel beads and carrying out reverse transcription PCR and / or PCR amplification to provide a composition comprising DNA or cDNA products, wherein the hydrogel beads are melted before or after step (c); and

[0078] (e) sequencing DNA or cDNA products of step (d).

[0079] In some embodiments, the hydrogel beads are re-encapsulated into droplets in an oil emulsion with PCR reagents and primers after washing. In some embodiments, barcoding elements are also included when the hydrogel beads are re-encapsulated.

[0080] Barcode sequences

[0081] In some embodiments, barcode elements (oligonucleotides / sequences) are included in the droplets when the hydrogel beads are re-encapsulated. The barcode elements may be incorporated into the DNA or cDNA products during step (d), as described above.

[0082] The barcode sequences may identify cellular analytes that were captured within the same hydrogel bead (e.g. cellular analytes originating from the same single cell), and distinguish cellular analytes that were captured within different hydrogel beads (e.g. cellular analytes originating from different single cells). In some cases, other barcode sequences, sometimes referred to as unique molecular identifier sequences (UMIs), can also be used to distinguish individual captured analytes (amplicons) or the amplification products thereof.

[0083] Barcode elements are typically short oligonucleotides comprising short, diverse nucleotide sequences. A pool of barcode elements having diverse nucleotide sequences could be generated using, for example, typical split and pool or degenerative synthesis methods, as are well known in the art. For example, 12 cycles of split-and-pool synthesis results in 412(16,777,216) possible barcode sequences. A typical barcode sequence may be at least 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length, for example, about 10 to 20, or 12 to 18, or 14 to 16 nucleotides in length. In some embodiments, the barcode sequences may be 15 nucleotides in length.

[0084] In some embodiments, nucleotides including barcoded sequences (e.g. 100 bp nucleotides comprising 15 bp barcode sequences) are re-encapsulated into droplets with the hydrogel beads at a limiting dilution. The use of a limiting dilution controls the probability of each droplet containing one or more barcode sequences. The limiting dilution can be adjusted to reach the desired compromise between sample loss (too many droplets comprising a hydrogel bead but no barcode sequences) and increased complexity / reduced accuracy in assigning barcoded sequences to the correct cells (too many droplets containing a hydrogel bead and multiple or too many different barcode sequences). For example, for applications of the method that involve paired sequencing of BCRs and TCRs, the barcode sequences that are re-encapsulated with each hydrogel bead may be linked to the ‘beta’ transcript. After PCR amplification and sequencing, the PCR replicates can be collapsed based on the barcode sequences and the number of cells that express the same beta can be estimated. Hence, the barcodes act as a marker of expansion. Hence, in this example, the number of different barcodes associated with a given linked TCR or BCR transcript provides an indication of the number of cells expressing the transcript, and therefore can be used to identify expanded TCR and BCR sequences. This is useful when using the methods of the invention in a screening method for immune receptors or antibodies.

[0085] For applications that involve pairing analytes expressed by single cells, it is necessary to add the barcode sequences at the re-encapsulation stage. However, for other applications, barcode sequences can also or alternatively be used during the initial analyte capture step / first encapsulation step, e.g. by incorporating barcode sequences (and / or UMIs) into the initial cellular analyte capture agent oligonucleotides. In some embodiments, the barcode sequences are added during step (c) of the method of single cell sequencing. However, the barcode sequences may be added during step (a) or between steps (d) and (e).

[0086] PCR

[0087] The library generation / single cell sequencing methods of the invention comprise a step (d) of reverse transcription PCR and / or PCR amplification to provide a composition comprising DNA or cDNA products.

[0088] In some embodiments, step (d) is carried out:

[0089] (i) within the re-encapsulated droplets (e.g. after the washing step described above); and / or

[0090] (ii) after breaking the droplets and combining the aqueous phase from the droplets (i.e. in bulk).

[0091] Typically, the melting temperature of the hydrogel bead is lower than the temperatures used for the PCR step. Hence, in some embodiments, the beads are maintained below the melting temperature of the hydrogel beads up to the PCR step. The hydrogel beads melt at the start of the PCR step, releasing the cellular analytes, bringing them into contact with the PCR reagents and allowing the PCR reaction to take place.

[0092] Carrying out the initial PCR reaction in-drop allows for the linking of paired analytes, such as the alpha and beta chains of TCRs or the heavy and light chains of BCRs, during the PCR reaction. For these types of application that involve linking paired analytes during the PCR stage it is important that the barcode sequences are added after the initial analyte capture (encapsulation and hydrogel formation) and before the PCR reaction (re-encapsulation of the hydrogel beads) during which the barcode sequences are linked to the relevant analytes.

[0093] Following reverse transcription and / or overlap extension, a nested PCR can be performed to amplify sequences of interest (e.g. TCR or BCR sequences), prior to sequencing. Even if the original reverse transcription and / or overlap extension is carried out in-drop, the nested PCR may be performed after breaking the second emulsion / in bulk because by this stage the barcodes have been linked to the analytes and, in appropriate embodiments, the target analytes have been paired.

[0094] Standard PCR reagents and methods can be used in the method of the invention. In some embodiments, the PCR primers are specific to at least one transcript of interest.

[0095] Immune receptors and antibodies

[0096] B cells and T cells each express immune receptors; B-cell receptors (BCRs) and T-cell receptors (TCRs). Both types of receptor consist of two polypeptide chains. BCRs comprise a heavy chain and a light chain, while TCRs comprise an alpha chain and a beta chain. Antibodies comprise a VH region and a VL region. Each polypeptide chain or VH / VL region comprises a constant region and a variable region. The variable regions result from recombination and end joint rearrangement of gene fragments on the chromosome of a B or T cell. In B cells, additional diversification of variable regions occurs by somatic hypermutation. The variable regions of both chains provide specificity for a given target.

[0097] Accordingly, in order to identify and study a given immune receptor or antibody, the sequences of both polypeptide chains must be known. One method of identifying which two polypeptide chains form a receptor and / or antibody is to use single cell sequencing, as each immune cell will express a pair of separate, unique transcripts. By linking the transcripts present in a single cell together, the complete sequence information for a given immune receptor or antibody can be preserved in downstream sequencing steps. In some embodiments, the at least one transcript of interest encodes a beta chain of a T cell receptor or a heavy chain of a B cell receptor.

[0098] In some embodiments, step (d) comprises overlap-extension PCR to link two of the captured RNA transcripts into a single cDNA product. In some embodiments, the two RNA transcripts encode:

[0099] (i) an alpha chain and a beta chain of a T cell receptor;

[0100] (ii) a heavy chain and a light chain of a B cell receptor; or

[0101] (iii) a VH region and a VL region of an antibody.

[0102] By linking the two RNA transcripts using overlap extension PCR at the same time as the barcode is introduced, the method allows for paired sequencing of the linked transcripts thereby allowing for not only the identification of pairs of polypeptide chains that comprise immune receptors, but also a measure of the expansion of said immune receptors. The methods of the invention therefore allow for the repertoire of immune receptors and antibodies in an individual organism or population of cells to be determined in a high throughput manner.

[0103] However, the method is not limited to use on immune receptors and antibodies, and can be used to study any two linked transcripts of interest. For example, the present method can be used to study gene co-expression patterns in different cell populations, for example in cancer cells.

[0104] Sequencing

[0105] Any suitable sequencing method may be used in the method of the invention. In some embodiments, the droplets are pooled before sequencing. In some embodiments, the step of sequencing further comprises a step of library preparation.

[0106] Hydrogel Beads

[0107] The present invention provides a hydrogel bead comprising at least one poly(dT) bead. As described above, the use of these beads in a method of single cell sequencing allows for an increase in throughput and decrease in the cost of sequencing methods, for example single cell sequencing methods to identify linked TCR and BCR sequences. The hydrogel bead may be an agarose hydrogel bead. Using a method as described herein, agarose may typically form a porous structure with a bead diameter of around 20 to 40 micrometres. Poly(dT) beads typically have a diameter of approximately 1-2 micrometres. The Poly(dT) beads are physically trapped within the pores of the hydrogel matrix. Advantageously, this avoids the need to covalently attach the beads to the hydrogel. The design allows for the incorporation of multiple poly(dT) beads within a single hydrogel bead, enhancing the capture efficiency of RNA molecules. Typically, around 10 to 100, or 20-50, or 30 to 40, or around 35 beads may be trapped within a single hydrogel bead.

[0108] The design of the hydrogel beads allows for capture of the cellular analytes without covalently attaching the capture elements to the hydrogel bead. Furthermore, in single cell sequencing applications, the hydrogel beads keep all the cellular analytes from a single cell together in a single droplet, allowing free barcodes to be added to the droplets, for example at a limiting dilution. This allows for both quantitative analysis of transcripts of interest and pairing of specific transcripts of interest. Furthermore, the barcode sequences do not need to be supplied attached to the cellular analytes capture agents, reducing the cost of the method.

[0109] The present invention also provides the use of the hydrogel bead in a method of the invention, as described herein.

[0110] Compositions and Kits

[0111] The present invention provides a composition comprising a hydrogel, poly(dT) beads and lysis buffer, as described elsewhere herein. In some embodiments, the hydrogel is molten hydrogel. Such a composition may form the second aqueous liquid phase in the methods of the invention.

[0112] The invention also provides a kit comprising the same elements, each provided in a separate container, or in any combination. The kit may further comprise a chip and / or device as described elsewhere herein and / or instructions for use in a method of the invention.

[0113] Devices

[0114] The present invention provides a device configured to perform a method according to the invention. The device may comprise means for storage and injection of the aqueous phases as described herein. The device may comprise means for storing an oil and allowing the oil to flow such that the aqueous phases may be injected thereto. The device may comprise a heating or temperature control element configures to facilitate melting and cooling of hydrogel in the device. For example, a tube-heater and slide heater may be used. The heating device may also be configured to provide variable temperatures suitable for carrying out a PCR reaction in the device, particularly in-drop PCR. The device may be suitable for use with a chip according to the invention, as described elsewhere herein.

[0115] Chips

[0116] The present invention provides chips configured for carrying out a method according to the invention. An example of such a chip is shown in Figure 5.

[0117] General definitions

[0118] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clear dictates otherwise. The term "and / or" as used in this application includes any and all combinations of one or more related listed items. The term “comprising” (means including but not limited to) specifically discloses / includes equivalent embodiments “consisting of’ (means limited to) or “consisting essentially of’ (means comprises no additional components that materially affect the essential characteristics of the subject matter). Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein. The term "about" as used herein generally means in quantitative terms plus or minus 10%, unless otherwise indicated.

[0119] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the technical field of this application. The terminology used in the description of this application is only for the purpose of describing specific embodiments, not to limit the scope of the invention as defined in the claims. The experimental methods without specific conditions in the following examples generally follow conventional conditions or the conditions recommended by the manufacturer. The common chemical reagents used in the examples are commercially available products. The terms “protein” and “polypeptide” are used interchangeably herein, and are intended to refer to a polymeric chain of amino acids of any length.

[0120] The terms “nucleic acid molecule”, “polynucleotide” and “nucleotide sequence” are intended to refer to a polymeric chain of any length of nucleotides, including deoxyribonucleotides, ribonucleotides, or analogues thereof. For example, the nucleic acid molecule, polynucleotide or nucleotide sequence may comprise DNA (deoxyribonucleotides) or RNA (ribonucleotides). The nucleic acid molecule, polynucleotide or nucleotide sequence may consist of DNA. The nucleic acid molecule, polynucleotide or nucleotide sequence may be mRNA. Since the nucleic acid molecule, polynucleotide or nucleotide sequence may comprise RNA or DNA, all references to T (thymine) nucleotides may be replaced with U (uracil).

[0121] Examples

[0122] Example 1: Exemplary protocol for single cell sequencing

[0123] Bead Composition and Properties

[0124] An RNA-capturing hydrogel bead was formed comprised primarily of Ultra-low Gelling Temperature Agarose (#A5030). This material forms a porous structure with a bead diameter ranging from 20 to 40 micrometers. Poly(dT) beads (NEB #S1419S) with a diameter of approximately 1-2 micrometers are physically trapped within the pores of the LMP-agarose matrix. The design allows for the incorporation of multiple poly(dT) beads within a single agarose bead, enhancing the capture efficiency of RNA molecules. Around 35 beads are trapped within a single agarose bead in the method disclosed. Internal capture elements also reduce likelihood of RNA contamination and assay noise. Demonstration of successful poly- A RNA capture shown in Fig. 4 using a poly-A fluorescent probe (FAM).

[0125] Description of Method

[0126] A single-step microfluidic process facilitates the simultaneous creation of beads and single-cell RNA capture. The process involves a mixture of 1.5-2% Ultra-low Gelling Temperature agarose and paramagnetic poly(dT) beads (5mg / ml) dissolved in 2x lysis buffer (200mM Tris (pH7.5), 500mM LiCl, 20mM EDTA (pH8.0), 1% LDS, lOmM DTT). Mixture is kept molten during infusion through use of a bespoke tube-heater and slide heater (tube heater at 60 °C, slide heater at 45 °C). Mixture is infused into microfluidic device alongside cell suspension (density matched with 21% Optiprep), at a concentration of 104 cells / pl. Up to 2M cells can be infused in 1 hour. A 1% solution of 008-FluoroSurfactant (Ran Biotechnologies) in 3M Novec 7500 Engineered Fluid was chosen as the continuous phase. Resulting droplet emulsions (~30um in diameter) are cooled at 4 °C to form solid agarose beads. After cooling droplets are broken through addition of 1H,1H,2H,2H-Perfluoro-1- octanol and beads are washed to remove lysates and resuspended in PCR reagents.

[0127] A second microfluidic step is used to re-encapsulate beads into droplets and allow in-drop RT-PCR. PCR reaction is designed to link two transcripts together into one DNA amplicon (alpha / beta chain of T cell receptor or heavy / light of B cell receptor) via overlap-extension PCR. PCR reagents include a barcode oligonucleotide at limiting dilution (IpM) which is amplified during reaction and links to either the beta or heavy chain. Surfactants are used to prevent coalescence during PCR (Tween or Igepal). Emulsion is collected into 200pl PCR tubes and thermocycled using the following conditions. BCR: 30 min. at 48°C, 2 min. at 94°C, 4 cycles of 30 s. at 94°C, 30 s. at 50°C and 2 min. at 68°C; 4 cycles of 30 s. at 94°C, 30 s. at 55°C and 2 min. at 68°C; 22 cycles of 30 s. at 94°C, 30 s. at 60°C and 2 min. at 68°C; final extension of 68°C for 7 min. TCR: 30 min. at 48°C, 1 min. at 94°C, 35 cycles of 15 s. at 94°C, 30 s. at 60°C and 1 min. at 68°C; final extension of 68°C for 5 min.

[0128] After PCR, droplets are broken through addition of Perfluoro- 1 -octanol. A nested PCR further amplifies desired product before library preparation and sequencing. One amplicon carries paired receptor information (alpha-beta / heavy-light), whilst the other is a barcoded signal (barcode-beta, barcode-heavy).

[0129] Adaptability

[0130] The method is adaptable to quantify the co-expression of any two transcripts of interest in various cell types (for example, linking a viral gene or cancer gene with a host immune receptor gene), offering a versatile tool for single-cell transcriptome analysis.

[0131] Experimental Data

[0132] The method above was used to pair BCR / TCR sequences in human cell line mixes which ascertain error rate of invention (ratios of 90%-10% and 50%-50%, Figure. 6). These revealed an error rate ranging from 2% to 10%, similar to that of other single-cell microfluidic platforms; 10X Genomics reporting an 8% error rate (at 10,000 cells), Drop-seq 0.36-11.3% (at 12.5-100 cells / l), InDrops 4%, and Seq-Well 1.6%. Sensitivity of our platform was confirmed up to 1% through cell-line spike-in experiments (3,000 cells, Figure. 6) Technology has also been applied to heathy donors and clinical cohorts to identify BCR / TCRs of interest (see Table 1).

[0133]

[0134] Example 2: Comparison with Dekosky method

[0135] Multiple beta chains to an alpha chain are rare, and estimated to be only 1% of the repertoire. Using this knowledge, a “fidelity score'' can measure the efficiency of RNA capture and the overall single cell resolution. If TCR / BCR data shows many beta chains to an alpha chain this suggests that the method has poor single-cell resolution. 100

[0136] Where:

[0137] • Fidelity(CDR3 A, BJ) represents the fidelity of a pairing for a specific alpha chain CDR3 and heavy J gene combination.

[0138] • Frequency(CDR3A, BJ) is the number of CDR3 -alpha / beta J gene pairs for the specific combination.

[0139] • Total(CDR3A) is the total frequency of pairings with a specific light chain CDR3.

[0140] This score is dependent upon:

[0141] • Efficiency of initial RNA capture

[0142] • Overall single cell resolution

[0143] • The higher this score, the better the single cell resolution of the assay.

[0144] Applying this measure to the only other high-throughput BCR / TCR assay (Dekosky) we see very poor fidelity scores (see Fig. 7A). Agarose bead method reproducibly exhibits very high-fidelity scores demonstrating much improved RNA-capture and ability to sustain single cell resolution (see Fig. 7B).

[0145] The agarose bead method reproducibly achieves high-fidelity scores. Four separate experiments on T cells from healthy donors and their resulting fidelity distributions are shown in Fig. 7C.

Claims

CLAIMS1. A method of capturing cellular analytes, the method comprising the steps of:(a) forming an emulsion by dispersing a first aqueous fluid phase comprising a suspension of cells and a second aqueous fluid phase comprising lysis buffer, a molten hydrogel and cellular analyte capture agents into a flowing oil phase simultaneously, such that the aqueous phases form droplets within the flowing oil; and(b) cooling the droplets such that hydrogel beads are formed within the droplets, the hydrogel beads being porous and comprising cellular analytes from the cell lysate captured by the cellular analyte capture agents within the pores of the hydrogel beads, wherein the cellular analyte capture agents are not covalently attached to the hydrogel bead.

2. The method of claim 1, wherein the cellular analytes are DNA, RNA, and / or polypeptides, optionally wherein the cellular analytes are mRNA.

3. The method of claim 1 or claim 2, wherein the cellular analyte capture agents comprise beads comprising oligonucleotides which bind to the cellular analytes, optionally wherein the cellular analyte capture agents are poly(dT) beads.

4. The method of any one of the preceding claims, wherein the cellular analyte capture agents or the beads comprising the oligonucleotides are between about 0.5 and 5 pm in diameter.

5. The method of any one of claims 1-4, wherein the majority of the droplets, optionally at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% of the droplets, comprise cell lysate from a single cell and / or a single agarose hydrogel bead.

6. The method of any one of claims 1- 5, wherein the cooling step comprises cooling at between 0 and 10 °C, between 2 and 6 °C, or between 3 and 5 °C, optionally wherein the step of cooling comprises cooling at around 4°C.

7. The method of any one of claims 1-6, wherein the method further comprises breaking the droplets, washing the hydrogel beads, and re-encapsulating the hydrogel beads into droplets in an oil emulsion after washing.

258. The method of claim 7, wherein the majority of the re-encapsulated droplets, optionally at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% of the re-encapsulated droplets, comprise a single hydrogel bead.

9. A method of single cell sequencing, comprising capturing cellular analytes in a hydrogel bead according to the method of any one of claims 1-8.

10. The method of claim 9, wherein the method comprises the steps of:(a) capturing cellular analytes on cellular analyte capture agents within hydrogel beads using a method according to any one of claims 1 to 7, wherein the cellular analytes are DNA or RNA;(b) breaking the emulsion and washing the hydrogel beads to remove excess cell lysate not captured within the agarose beads;(c) mixing the hydrogel beads with PCR reagents and primers;(d) carrying out reverse transcription PCR and / or PCR amplification to provide a composition comprising DNA or cDNA products;(e) sequencing DNA or cDNA products of step (d).

11. The method of claim 10, wherein the hydrogel beads are re-encapsulated into droplets in an oil emulsion with PCR reagents and primers after washing.

12. The method of claim 11, wherein the PCR reagents include barcode sequences that are linked to the DNA or cDNA products during step (d), optionally wherein the barcode sequences are encapsulated with the hydrogel beads at a limiting dilution.

13. The method of claim 11 or claim 12, wherein step (d) is carried out:(i) within the re-encapsulated droplets; and / or(ii) after breaking the droplets and combining the aqueous phase from the droplets.

14. The method of any one of claims 11-13, wherein at least one set of PCR primers is specific to at least one transcript of interest.

15. The method of claim 14, wherein the at least one transcript of interest encodes a beta chain of a T cell receptor or a heavy chain of a B cell receptor.

16. The method of any one of claims 10-15, wherein step (d) comprises overlap-extension PCR to link two of the captured RNA transcripts into a single cDNA product.

17. The method of claim 16, wherein the two RNA transcripts encode:(i) an alpha chain and a beta chain of a T cell receptor;(ii) a heavy chain and a light chain of a B cell receptor; or(iii) a VH region and a VL region of an antibody.

18. The method of any one of claims 10-17, wherein step (e) further comprises a step of library preparation.

19. A hydrogel bead comprising at least one poly(dT) bead.

20. Use of the hydrogel bead of claim 19 in a method of single cell sequencing.

21. A composition or kit comprising a hydrogel, poly(dT) beads and lysis buffer, optionally wherein the hydrogel is molten hydrogel.

22. A kit comprising:(i) a hydrogel;(ii) poly(dT) beads; and(iii) lysis buffer.

23. A device configured to perform the method of any one of claims 1-18.

24. A chip configured for carrying out the method of any one of claims 1-18.

25. The method of any one of claims 1-18, hydrogel bead of claim 19, use of claim 20, composition of claim 21, or kit of claim 22, wherein the hydrogel is agarose.28

Images