Hydrogel microspheres for single cell sequencing as well as preparation method and application of hydrogel microspheres

Abstract

The invention discloses hydrogel microspheres for single cell sequencing as well as a preparation method and application of the hydrogel microspheres. The hydrogel microsphere is a PEGDA gel microsphere with a porous structure, and a plurality of oligonucleotide fragments carrying tag sequences are connected to the surface and the interior of the porous structure of the hydrogel microsphere; the gel beads are prepared from a water phase and an oil phase through a droplet generation device under ultraviolet catalysis, the oil phase comprises droplet generation oil added with TEMED, and the water phase comprises an acrylamide monomer, PEGDA, ammonium persulfate, ethylene modified oligo, a TBSET buffer solution and water; and oligonucleotide is connected to oligo, so that the hydrogel microspheres are formed. The hydrogel microspheres cannot be dissolved in water-in-oil liquid drops and cannot form high-molecular polymer monomers, so that the inhibition effect of the high-molecular polymer monomers on reverse transcription reaction is avoided, the reverse transcription efficiency is improved, and the gene detection rate is further improved; and the preparation time of the gel microspheres is short, and the production is more convenient and quicker.

Description

Technical Field

[0001] The present application relates to the field of single-cell sequencing technology, and in particular to a hydrogel microsphere for single-cell sequencing, and a preparation method and application thereof. Background Art

[0002] Single-cell sequencing technologies are currently divided into two main categories: one is based on oil-in-water droplet platforms, with 10×genomics leading the pack; the other is based on micro-pit capture platforms, with BD Rhapsody being a prominent example. In terms of market share, 10×genomics holds a commanding lead. Oil-in-water single-cell platforms are user-friendly and easy to use. They utilize flexible hydrogel microspheres as oligo carriers, improving cell capture efficiency and boosting gene detection rates. However, the core reagents of oil-in-water single-cell platforms are expensive, resulting in high costs, and the hydrogel microsphere production process is complex and time-consuming. Hydrogel microspheres typically require oven drying for more than 10 hours. Furthermore, within the oil-in-water droplets, the soluble hydrogel is dissolved by reducing agents, producing large polymers. These polymers have inhibitory effects, interfering with biochemical reactions within the droplets, reducing cDNA yield and, consequently, gene detection rates.

[0003] Current platforms for single-cell encapsulation in microdroplets employ either dissolvable polyacrylamide hydrogel beads or photocleavage-based primer release. Both approaches aim to fully release the oligo primers carried by the microspheres, allowing the released molecular tags to fully bind to the mRNA within the droplets and complete the reverse transcription reaction. However, both dissolvable hydrogel microspheres and photocleavage-based hydrogel microspheres suffer from complex preparation processes, high production costs, batch-to-batch instability, and difficulties in mass production.

[0004] In short, how to improve the preparation efficiency of hydrogel microspheres, further improve the reverse transcription efficiency, and increase the gene detection rate remains the research focus and difficulty in the field of single-cell sequencing technology. Summary of the Invention

[0005] The purpose of this application is to provide a new hydrogel microsphere for single-cell sequencing and its preparation method and application.

[0006] This application adopts the following technical solutions:

[0007] The first aspect of the present application discloses a hydrogel microsphere for single-cell sequencing, which has a structure of poly(ethylene glycol) diacrylate (PEGDA) gel microspheres with a porous structure, and a plurality of oligonucleotide fragments carrying tag sequences are connected on the surface of the poly(ethylene glycol) diacrylate gel microspheres and in their porous structure; the poly(ethylene glycol) diacrylate gel microspheres are prepared by a droplet generation device under ultraviolet catalysis from an aqueous phase solution and an oil phase solution, wherein the oil phase solution includes a droplet generation oil added with tetramethylethylenediamine, and the aqueous phase solution includes acrylamide monomer, poly(ethylene glycol) diacrylate, ammonium persulfate, ethylene-modified oligo, TBSET buffer and water; the aqueous phase solution and the oil phase solution generate poly(ethylene glycol) diacrylate gel microspheres with a porous structure, and a plurality of oligos are carried on the surface and in the porous structure of the poly(ethylene glycol) diacrylate gel microspheres, and the desired oligonucleotides are then connected to the oligos through a ligation reaction to form the hydrogel microspheres of the present application.

[0008] It should be noted that the hydrogel microspheres of the present application do not dissolve in the water-in-oil microdroplets and will not form high-molecular polymer monomers, thereby not inhibiting the reverse transcription process, improving the reverse transcription efficiency, and helping to improve the gene detection rate.

[0009] Preferably, each poly(ethylene glycol) diacrylate gel microbead is coupled to at least one million oligonucleotide fragments.

[0010] It should be noted that the hydrogel microspheres of the present application can simply and conveniently connect millions of oligonucleotide fragments, thereby meeting the use requirements of single-cell transcript capture.

[0011] Preferably, each oligonucleotide fragment consists of oligo, F2 sequence, F3 sequence and F4 sequence; wherein oligo is a universal oligonucleotide sequence for poly (ethylene glycol) diacrylate gel microbeads; F2 sequence is a random sequence of 4-15 bp in length; F3 sequence is composed of the first linker sequence, (N) x , and the second connecting sequence. The length of the first connecting sequence and the second connecting sequence are both 4-8bp. The first connecting sequence is used to connect with the oligo, and the second connecting sequence is used to connect with the F4 sequence. The F4 sequence is composed of the third connecting sequence, (N) from the 5' end to the 3' end. x , T tail, the length of the third linker sequence is 4-8bp, used to connect with the F3 sequence, the length of the T tail is 20-50bp; in the F3 sequence and F4 sequence, x is 4-15, N is any base of A, G, C, T, (N) x Indicates a random sequence of 4-15 bp in length.

[0012] Preferably, the oligo is the sequence shown in SEQ ID NO.1;

[0013] SEQ ID NO. 1: 5'-AATGATACGGCGACCACCGAGATCTACACgatct-3'.

[0014] Preferably, hydrogel microspheres in the same batch have the same first linking sequence, second linking sequence and third linking sequence.

[0015] Preferably, the first linking sequence is tgtcac, the second linking sequence is agatc, and the third linking sequence is atatcg.

[0016] The second aspect of the present application discloses the use of the hydrogel microspheres of the present application in preparing single cell detection reagents.

[0017] The third aspect of the present application discloses a single cell detection reagent, comprising the hydrogel microspheres of the present application.

[0018] The fourth aspect of the present application discloses a method for preparing the hydrogel microspheres of the present application, comprising the following steps:

[0019] The oil phase solution and the aqueous phase solution are simultaneously injected into a droplet generation device and irradiated with ultraviolet light. In the droplet generation device, the aqueous phase is broken up into uniform small droplets by the oil phase through shear force, and then cross-linked by ultraviolet light to form poly(ethylene glycol) diacrylate gel microbeads, which are collected for later use;

[0020] The oil phase in the collection tube is removed, and the generated poly (ethylene glycol) diacrylate gel microbeads are retained and added with TBSET solution; the PFO solution is then added, mixed, centrifuged, and the bottom PFO solution is removed, and the addition of PFO solution, mixing, centrifugation, and the removal of the bottom PFO solution are repeated at least once; then the span80 n-hexane solution is added, mixed, centrifuged, and the top span80 n-hexane solution is removed, and the addition of span80 n-hexane solution, mixing, centrifugation, and the removal of the top span80 n-hexane solution are repeated at least once; the TBSET solution is added, mixed, centrifuged, and the top TBSET solution is removed, and the addition of TBSET solution, mixing, centrifugation, and the removal of the top TBSET solution are repeated at least three times; and then filtered with a filter to remove oversized beads and impurities to obtain uniform poly (ethylene glycol) diacrylate gel microbeads;

[0021] The desired oligonucleotide is connected to the oligo using a ligase to form the hydrogel microspheres of the present application.

[0022] It should be noted that the gel beads generated by the preparation method of the present application have similar loose porous properties to the currently commonly used soluble PAGE hydrogel beads, so that the mRNA molecules released by cell lysis can be captured by oligonucleotide fragments on the surface and inside of the PEGDA gel beads. At the same time, the gel beads prepared by the present application will not be dissolved in the oil-in-water droplets, thereby avoiding the inhibitory effect of the release of high-molecular polymer monomers on the reverse transcription reaction like the lysis of gel beads made of other materials, thereby improving the reverse transcription efficiency. And most importantly, the preparation method of the present application can complete the preparation of PEGDA hydrogel microspheres within 30 minutes, and then the subsequent nucleic acid coupling can be carried out immediately. Compared with hydrogel microspheres made of other materials, the overall preparation time is greatly shortened, which is more convenient and quick.

[0023] Preferably, in the preparation method of the present application, a ligase is used to connect the desired oligonucleotide to the oligo, specifically comprising performing ligation reactions of the F2 sequence, the F3 sequence, and the F4 sequence, respectively. After the ligation reaction is completed, the supernatant and the complementary chain are removed to obtain the hydrogel microspheres of the present application having oligonucleotide fragments carrying the tag sequence connected to the surface and the porous structure thereof.

[0024] The beneficial effects of this application are:

[0025] The hydrogel microspheres of the present application have a loose, porous structure, allowing mRNA molecules released by cell lysis to be captured by oligonucleotide fragments on the surface and within the hydrogel microspheres. Furthermore, the hydrogel microspheres of the present application do not dissolve within the water-in-oil droplets and do not form polymer monomers, thereby avoiding the inhibitory effect of polymer monomers on the reverse transcription reaction, improving reverse transcription efficiency, and thereby increasing gene detection rates. The preparation method of the hydrogel microspheres of the present application has a short generation time of poly(ethylene glycol) diacrylate gel microbeads, greatly shortening the overall preparation time and making it more convenient and quick. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a real-time microscope observation result at the cross-section of the droplet generation chip in the embodiment of the present application;

[0027] Figure 2 This is a diagram showing the real-time observation results of micro-droplets in the flow channel of the droplet generation chip under a microscope in an embodiment of the present application;

[0028] Figure 3 This is a photo of PEGDA gel microspheres collected in a collection tube in an embodiment of the present application;

[0029] Figure 4 1 is a microscopic observation result of the PEGDA gel microbeads washed and recovered in the embodiment of the present application;

[0030] Figure 5This is a microscopic observation result of PEGDA gel microbeads connected with complete oligonucleotide fragments in the examples of the present application;

[0031] Figure 6 This is a diagram showing the results of fluorescent probe hybridization detection of PEGDA gel microbeads connected to complete oligonucleotide fragments in the examples of this application. DETAILED DESCRIPTION

[0032] The existing method for preparing gel beads by encapsulating single cells in microdroplets has complex processes, high production costs, instability between batches, and difficulty in mass production. In order to solve these problems, the present application has redeveloped a rapidly generated poly (ethylene glycol) diacrylate (PEGDA) gel microbeads for single-cell sequencing, and coupled millions of oligonucleotides to each microbead through a ligation reaction for capturing transcripts in the cell, and ensured that each microbead carries a unique tag sequence for distinguishing transcripts from different cells. In addition, this gel microbead will not dissolve and will not interfere with the subsequent reverse transcription reaction in the microdroplets, thereby improving the capture efficiency of intracellular transcripts. Our gel bead preparation process is simple to operate, low-cost, and suitable for large-scale production. The prepared functionalized gel beads can be widely used in application fields such as single-cell transcriptomics, epigenetics, CRISPR screening, and single-cell multi-omics.

[0033] The present invention is further described in detail below by means of specific examples. The following examples are only provided to further illustrate the present invention and should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0034] Example

[0035] 1. Generation of PEGDA gel microbeads

[0036] 1. For oil phase preparation, add 10 μL of tetramethylethylenediamine (TEMED) to 2.5 mL of droplet generation oil (Bio-Rad, Droplet Generation Oil for Probes #1863005), vortex to mix, and set aside.

[0037] 2. Add the prepared oil phase into the syringe, connect the syringe needle to the catheter, and slowly push the syringe to allow the droplet-generated oil to reach the top of the catheter.

[0038] 3. Prepare the aqueous solution of PEGDA-modified primers according to Table 1, vortex to mix, and briefly centrifuge for 5 seconds.

[0039] Table 1 Aqueous phase preparation scheme for gel microbead generation

[0040]

[0041]

[0042] The oligo in this example is the sequence shown in SEQ ID NO.1;

[0043] SEQ ID NO. 1: 5'-AATGATACGGCGACCACCGAGATCTACACgatct-3'.

[0044] Ethylene-modified oligos were synthesized by Sangon Biotechnology. 1× TBSET buffer was purchased from Shanghai Panchao Biotechnology, catalog number PCDBE076.

[0045] 4. Add the prepared aqueous phase into the syringe (pre-loaded with 200 μL HEF-7500, filler), connect the syringe needle to the catheter, and slowly push the syringe until the aqueous phase reaches the top of the catheter.

[0046] 5. PEGDA gel microbeads were prepared using a single-cell microdroplet generator equipped with a UV lamp (purchased from Shanghai Pengzan Biotechnology, model DG-01). A 1.5 mL collection tube was covered with 200 μL of light mineral oil (purchased from Bio-Rad, cat. no. 1632129). The oil phase flow rate was set to 800 μL / h, and the aqueous phase flow rate to 400 μL / h. The UV lamp was turned on at the collection tube or its outlet, and the aqueous and oil phase microinjection pumps were simultaneously activated to generate droplets.

[0047] The real-time microscope observation results at the cross section of the droplet generation chip are as follows: Figure 1 As shown, the real-time observation results of the micro-droplet microscope in the flow channel are as follows Figure 2 The generated PEGDA gel microspheres were collected in a collection tube as shown. Figure 3 shown.

[0048] Figure 1 and Figure 2 The results showed that PEGDA gel microbeads with uniform size were prepared in this example; Figure 3 In this example, the bottom layer is droplet formation oil, the middle layer is PEGDA gel microbeads, and the top layer is light mineral oil. In this example, the generated microdroplets are directly irradiated with UV light, and curing is completed as the generation progresses. This significantly reduces the curing time of the microspheres, allowing the preparation of PEGDA gel microbeads to be completed in half an hour, making it more convenient for batch production of microspheres.

[0049] 2. Cleaning and recovery of PEGDA gel microbeads

[0050] 1. Remove the mineral oil on the top of the collection tube and the oil generated by the droplet at the bottom, and add 500 μL of 1× TBSET buffer.

[0051] 2. Add 1 mL of 20% PFO solution (perfluoro-1-octanol, Merck, 647-42-7), vortex to mix, and centrifuge at 3000 g for 1 minute.

[0052] 3. Remove the PFO solution at the bottom, add 1 mL of 20% PFO solution again, vortex to mix, and centrifuge at 3000 g for 1 minute.

[0053] 4. Remove the PFO solution at the bottom, add 1 mL of 1% span80 n-hexane solution, vortex to mix, and centrifuge at 3000 g for 1 minute.

[0054] 5. Remove the top 1% Span80 hexane solution, add 1 mL of 1% Span80 hexane solution again, vortex to mix, and centrifuge at 3000 g for 1 minute.

[0055] 6. Remove the top 1% span80 hexane solution.

[0056] 7. Add 1 mL of 1× TBSET buffer, vortex to mix, and centrifuge at 3000 g for 1 minute. Remove the top of the TBSET buffer.

[0057] 8. Repeat step 7 4 times.

[0058] 9. Filter the beads with a 70 μm filter to remove oversized beads and impurities to obtain relatively uniform PEGDA gel microbeads.

[0059] The PEGDA gel microbeads were observed under a microscope. Figure 4 shown.

[0060] Figure 4 The results showed that PEGDA gel microbeads of uniform size were prepared in this example.

[0061] 3. Nucleic Acid Coupling of Diverse PEGDA Gel Beads

[0062] The PEGDA gel microbeads prepared above have oligos on their surface and in their porous structure. On this basis, different oligonucleotide fragments can be further connected as needed to obtain hydrogel microbeads for different single-cell sequencing. The specific connection reaction is as follows:

[0063] 1. Dilution of oligonucleotide primers: Centrifuge the primer tube at 4000 rpm for 30-60 seconds before opening the tube cap. Slowly open the tube cap and add an appropriate amount of double-distilled water to form a 100 μM solution. Cover the tube cap and vortex thoroughly to mix. Centrifuge briefly and set aside.

[0064] 2. Obtaining F2-F3 sequences: Dissolve 96 F2 sequences and 96 F3 sequences into 100 μM solutions according to the primer synthesis report. Add 40 μL each of F2 and F3 to a microplate. Add 20 μL of 5× DNA annealing buffer (Biyuntian, R0216). Heat the PCR instrument to 95°C and slowly anneal to 15°C to obtain 96 F2-F3 sequences.

[0065] 3. First step ligation reaction:

[0066] (1) Open the metal bath in advance, set the reaction temperature to 20°C, the rotation speed to 50 rpm, and the reaction time to 60 min, and then mix the reaction system as shown in Table 2 into a 2.0 mL centrifuge tube (96 tubes) for reaction. Among them, the F2-F3 sequence is the sequence that has been connected previously. This structure can be directly connected to the sequence on the gel bead.

[0067] Table 2 Configuration of the first step ligation reaction system

[0068] Components Volume (μL) 10×T4Ligasebuffer 100 T4Ligase 100 F2~F3 sequence 30 Beads with oligo sequences 300 <![CDATA[H2O]]> 470 total 1000

[0069] (2) Termination of the first ligation reaction: After the first ligation reaction is completed, use a fast centrifuge, set the speed to 4500 rpm, centrifuge for 2 minutes, and discard the supernatant; then add 1 mL of 10 mM Tris-HCl (pH 8.0), shake and mix, and centrifuge at 4500 rpm for 2 minutes; add 1 mL of 10 mM Tris-HCl (pH 8.0) solution, shake and mix, and centrifuge at 4500 rpm for 2 minutes, and discard the supernatant.

[0070] (3) Finally, dilute to 300 μL with 10 mM Tris-HCl (pH 8.0) and place in a 2.0 mL centrifuge tube.

[0071] After the above connection reaction, PEGDA gel microbeads with oligo~F2~F3 sequences connected in sequence are obtained.

[0072] 4. Second ligation reaction

[0073] (1) Add the reaction reagents listed in Table 3 below to a centrifuge tube containing PEGDA gel microbeads linked to the oligo~F2~F3 sequence. Open the metal bath in advance and set the reaction temperature to 20°C, the speed to 50 rpm, and the reaction time to 60 min. Then, mix the reaction mixture as shown in Table 3 and add it to the centrifuge tube containing PEGDA gel microbeads (96 types) for reaction.

[0074] Table 3 Reagent configuration for the second step ligation reaction

[0075] Components Volume (μL) 10×T4Ligasebuffer 100 T4Ligase 100 Tag primer F4 30 (96 types) <![CDATA[H2O]]> 470 total 1000

[0076] (2) Terminate the second-step ligation reaction: After the ligation reaction is completed, use a fast centrifuge, set the speed to 4500 rpm, centrifuge for 2 minutes, and discard the supernatant; then add 1 mL of 10 mM Tris-HCl (pH 8.0), shake and mix, and centrifuge at 4500 rpm for 2 minutes; add 1 mL of TET solution, shake and mix, and centrifuge at 4500 rpm for 2 minutes, and discard the supernatant.

[0077] (3) Finally, the volume was adjusted to 300 μL of 10 mM Tris-HCl (pH 8.0) in a 2.0 mL centrifuge tube.

[0078] After the above connection reaction, PEGDA gel microbeads with oligo, F2, F3, and F4 sequences connected in sequence are obtained.

[0079] In this case, 96 F2 primers, 96 F3 primers, and 96 F4 primers were designed for single-cell sequencing of human PBMC cells.

[0080] The F2 sequence is a random sequence with a length of 10 bp, that is, the sequence of F2 is SEQ ID NO. 5: NNNNNNNNNN.

[0081] The F3 sequence from 5' to 3' end consists of the first linker sequence, (N) 10 , the second connecting sequence, the first connecting sequence is tgtcac, the second connecting sequence is agatc, (N) 10 represents a 10 bp random sequence, that is, the F3 sequence from the 5' end to the 3' end is SEQ ID NO. 4: tgtcacNNNNNNNNNNagatc.

[0082] The F4 sequence from the 5' end to the 3' end consists of the third linker sequence, (N) 10 , T tail, the third connecting sequence is atatcg, the length of the T tail is 30bp, that is, the F4 sequence from 5' end to 3' end is SEQ ID NO.3: atatcgNNNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT.

[0083] The prepared PEGDA gel microbeads connected with oligo, F2, F3, and F4 sequences were observed under a microscope. Figure 5 shown. Figure 5 The results showed that PEGDA gel microbeads of uniform size were prepared in this example.

[0084] Fluorescent probe hybridization was used to detect PEGDA gel microbeads linked to oligo, F2, F3, and F4 sequences, qualitatively observing the coupling of the full-length molecular tag attached to the microbeads. Specifically, the fluorescent probe used in this example was the sequence shown in SEQ ID NO. 2, labeled with FAM fluorescence, and synthesized by Shanghai Bioengineering. SEQ ID NO. 2: 5'-AAAAAAAAAAAAAAAAAAAAAAAA-FAM-3'.

[0085] The detection method of the fluorescent probe is as follows: take 10 μL of PEGDA gel microbeads connected with the ligo, F2, F3, and F4 sequences, add 1 μL of 100 μM probe of SEQ ID NO. 2, incubate at room temperature for 10 minutes, wash the beads three times with 10 mM Tris-HCl (pH 8.0), and then place them under a fluorescence microscope to take pictures, first in the bright field, and then in the fluorescence field under the FAM channel.

[0086] Bright field Figure 5 As shown, the fluorescence field is Figure 6 As shown, the results show that the fluorescence brightness under the fluorescence field is relatively uniform and can correspond well to the bright field.

[0087] 4. Single-cell sequencing based on PEGDA gel beads

[0088] Single-cell sequencing of human PBMC cells was performed using the previously prepared PEGDA gel microbeads linked to oligo, F2, F3, and F4 sequences. For specific procedures, refer to the 10×Genomics, CG000315 instruction manual, except that the beads in the manual were replaced with PEGDA gel microbeads linked to oligo, F2, F3, and F4 sequences.

[0089] At the same time, the same single-cell sequencing was performed on the same amount of cells using existing PAGE microspheres (10×Genomics, 2000164) for comparison.

[0090] The statistics of the sequencing results of the PEGDA gel microbeads and PAGE microspheres in this example are shown in Table 4.

[0091] Table 4 Single cell sequencing results of different microspheres

[0092]

[0093]

[0094] The results in Table 4 show that with the same amount of sequencing data and roughly equivalent sequencing saturation, PEGDA gel microbeads obtained a higher median gene number and a higher median UMI, indicating that PEGDA microbeads are superior to PAGE gel microbeads in mRNA capture and reverse transcription within microdroplets.

[0095] The above content is a further detailed description of the present application in conjunction with specific implementation methods, and the specific implementation of the present application cannot be considered to be limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A hydrogel microsphere for single-cell sequencing, characterized by: The poly(ethylene glycol) diacrylate gel microbeads have a porous structure, and a plurality of oligonucleotide fragments carrying tag sequences are connected to the surface of the poly(ethylene glycol) diacrylate gel microbeads and the porous structure thereof; The poly(ethylene glycol) diacrylate gel microbeads are prepared from an aqueous solution and an oily solution by a droplet generation device under ultraviolet catalysis, wherein the oily solution comprises a droplet generation oil added with tetramethylethylenediamine, and the aqueous solution comprises acrylamide monomer, poly(ethylene glycol) diacrylate, ammonium persulfate, ethylene-modified oligo, TBSET buffer, and water; The aqueous solution and the oily solution generate poly(ethylene glycol) diacrylate gel microbeads with a porous structure, and a plurality of oligos are carried on the surface and in the porous structure. The desired oligonucleotides are then connected to the oligos through a ligation reaction to form the hydrogel microspheres.

2. The hydrogel microspheres according to claim 1, wherein: Each poly(ethylene glycol) diacrylate gel microbead is coupled to at least one million oligonucleotide fragments.

3. The hydrogel microspheres according to claim 1, wherein: Each oligonucleotide fragment consists of oligo, F2 sequence, F3 sequence and F4 sequence; Wherein, oligo is the oligonucleotide sequence universal for poly(ethylene glycol) diacrylate gel microbeads; The F2 sequence is a random sequence of 4-15 bp in length; The F3 sequence from 5' to 3' end consists of the first linker sequence, (N) x , the second connecting sequence, the length of the first connecting sequence and the second connecting sequence are both 4-8bp, the first connecting sequence is used to connect with the oligo, and the second connecting sequence is used to connect with the F4 sequence; The F4 sequence from the 5' end to the 3' end consists of the third linker sequence, (N) x , T tail, the length of the third linker sequence is 4-8 bp, used for connecting with the F3 sequence, and the length of the T tail is 20-50 bp; In the F3 and F4 sequences, x is 4-15, N is any base of A, G, C, or T, (N) x Indicates a random sequence of 4-15 bp in length.

4. The hydrogel microspheres according to claim 3, characterized in that: The oligo is the sequence shown in SEQ ID NO.1; SEQ ID NO. 1: 5'-AATGATACGGCGACCACCGAGATCTACACgatct-3'.

5. The hydrogel microspheres according to claim 3, characterized in that: The hydrogel microspheres in the same batch have the same first connection sequence, second connection sequence and third connection sequence.

6. The hydrogel microspheres according to claim 5, characterized in that: The first linking sequence is tgtcac, the second linking sequence is agatc, and the third linking sequence is atatcg.

7. Use of the hydrogel microspheres according to any one of claims 1 to 6 in the preparation of single cell detection reagents.

8. A single cell detection reagent, characterized in that: The hydrogel microspheres include those described in any one of claims 1 to 6.

9. The method for preparing the hydrogel microspheres according to any one of claims 1 to 6, characterized in that: The following steps are included: The oil phase solution and the aqueous phase solution are simultaneously injected into a droplet generation device and irradiated with ultraviolet light. In the droplet generation device, the aqueous phase is broken up into uniform small droplets by the oil phase through shear force, and then cross-linked by ultraviolet light to form poly(ethylene glycol) diacrylate gel microbeads, which are collected for later use; The oil phase in the collection tube was removed, the generated poly(ethylene glycol) diacrylate gel microbeads were retained, and TBSET solution was added; Then add PFO solution, mix, centrifuge, remove the bottom PFO solution, repeat adding PFO solution, mix, centrifuge, remove the bottom PFO solution at least once; then add span80 n-hexane solution, mix, centrifuge, remove the top span80 n-hexane solution, repeat adding span80 n-hexane solution, mix, centrifuge, remove the top span80 n-hexane solution at least once; add TBSET solution, mix, centrifuge, remove the top TBSET solution, repeat adding TBSET solution, mix, centrifuge, remove the top TBSET solution at least three times; Then, the mixture was filtered through a filter to remove oversized beads and impurities to obtain uniform poly(ethylene glycol) diacrylate gel microbeads; The desired oligonucleotide is connected to the oligo using a ligase to form the hydrogel microspheres.

10. The preparation method according to claim 9, characterized in that: The use of a ligase to connect the desired oligonucleotide to the oligo specifically includes performing ligation reactions on the F2 sequence, the F3 sequence, and the F4 sequence, respectively. After the ligation reaction is completed, the supernatant and the complementary chain are removed to obtain the hydrogel microspheres having oligonucleotide fragments carrying the tag sequence connected on the surface and in the porous structure thereof.

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