Bar code repeatable microsphere and preparation method and application thereof

Abstract

The invention discloses a barcode repeatable microsphere as well as a preparation method and application thereof, and relates to the field of biomedical detection and high-throughput single cell sequencing. The bar code repeatable microsphere disclosed by the invention comprises a microsphere, a universal primer probe and a bar code probe, wherein the bar code probe comprises a Nick region, a capture region, a cell bar code region and a molecular bar code region. According to the method, preparation of the barcode microspheres and repetition of barcodes are achieved at extremely low cost, operation is easy, the application field is wide, and the method can be used for detection of genomes, transcriptomes, proteomes, epigenomes, metabolomes and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection and high-throughput single-cell sequencing, and in particular to a preparation method of barcode repeatable microspheres and applications thereof. Background Art

[0002] Biomedical testing is a science that uses biomedical principles and methods to detect, measure and analyze organisms and their components through specific technical means. It involves a variety of principles, such as optics, electrochemistry, immunology, biology, etc., and plays a vital role in medical diagnosis, treatment monitoring, disease prevention, drug development, biomedical research and other fields. Biomedical testing involves many aspects, including gene detection, protein detection, metabolite detection, cell analysis, bioimaging, etc. Among them, single cell analysis (SCA) is a method for in-depth research on single cells, aiming to reveal the information of single cells at the genome, transcriptome, epigenome, proteome and other levels. Through single cell analysis, gene mutations and gene expression changes in single cells can be detected, which is helpful for early diagnosis of diseases. In oncology, single cell analysis can identify tumor cell subpopulations with different gene expression patterns, providing a basis for personalized treatment. In basic medical research, single cell analysis can be used to study biological processes such as stem cell differentiation and apoptosis, providing new ideas for regenerative medicine and disease treatment. Therefore, single-cell analysis plays an important role in biomedical testing, helping to improve the accuracy of disease diagnosis, achieve personalized medicine, and accelerate the process of drug development. With the continuous advancement of technology and the expansion of its application scope, single-cell analysis will play an even more important role in the biomedical field in the future.

[0003] Barcode microspheres play an important role in single-cell analysis, especially in high-throughput single-cell sequencing and analysis. Barcode microspheres are tiny spheres with unique barcode identifiers. The barcode probes on the microspheres usually include the following functional regions: universal primers, cell barcodes, molecular barcodes, and capture regions. The use of barcode microspheres enables the application of high-throughput sequencing technology at the single-cell level. Each cell is given a unique barcode, so that even in mixed samples, sequencing data from different cells can be accurately distinguished, greatly improving the efficiency and accuracy of single-cell sequencing.

[0004] At present, the barcode microspheres for high-throughput single-cell sequencing mainly include the following: 1) Macosko-2011-10 (V+) provided by ChemGenes, which is a common commercial barcode microsphere, usually used for high-throughput single-cell sequencing technology. It is made of resin material, and the surface is modified with oligonucleotides with poly-dT sequences for capturing and labeling mRNA, cell barcode sequences for distinguishing different cells, molecular barcode sequences for molecular quantification, and universal adapter sequences for downstream next-generation sequencing; 2) Gel microspheres (GEMs) from 10x Genomics, which are gel microspheres modified with nucleic acid probes. These nucleic acid probes also include poly-dT, cell barcodes, molecular barcodes, and universal adapters. In the use of GEMs, cells are restrictedly diluted to ensure that most GEMs do not contain cells, and most GEMs containing cells contain only single cells. Then, within the GEMs, the cells are lysed and the gel beads are dissolved, releasing a large number of barcoded nucleic acid probes that capture mRNA with a PolyA tail and are reverse transcribed to generate the first-strand cDNA with 10x Barcode and UMI information.

[0005] However, during the use of the above-mentioned Macosko-2011-10 (V+) microspheres, the nucleic acid probe with the barcode functional region will participate in the synthesis of cDNA during the reverse transcription process, resulting in the consumption of the barcode probe, which makes the microspheres unable to be reused. The gel microspheres with barcode probes from 10x Genomics will be dissolved and consumed during use and cannot be reused. At the same time, since the preparation of barcode microspheres requires professional equipment and complex processes, and high-throughput single-cell sequencing detection has extremely high quality requirements for microspheres and barcodes, otherwise it will affect the final sequencing results. This is why there is only one commercial barcode microsphere, Macosko-2011-10 (V+). On the one hand, this leads to the high cost of microspheres for high-throughput single-cell sequencing; on the other hand, the commercial barcode microspheres are of a single type and cannot meet the current multi-omics and multi-target detection requirements, which greatly limits the flexibility of sequencing technology applications. Therefore, it is of great significance to develop a barcode repeatable microsphere with a simple synthesis method and low cost for high-throughput single-cell analysis. Summary of the invention

[0006] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to develop a barcode reusable microsphere and its preparation method and application, aiming to solve the technical problems that the barcode microspheres prepared by the prior art are disposable and cannot be reused, and the preparation cost is high and the equipment used is complex.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention discloses a barcode repeatable microsphere, comprising a microsphere, a universal primer probe and a barcode probe;

[0009] The barcode probe is coupled to the microsphere, the universal primer probe can hybridize with the barcode probe, and the universal primer probe is used for PCR amplification;

[0010] The barcode probe includes a Nick region, a capture region, a cell barcode region and a molecular barcode region, wherein:

[0011] Nick region, used for SDA reaction;

[0012] A capture zone, used to capture the molecules to be detected;

[0013] Cell barcode region, used for cell identification;

[0014] The molecular barcode region is used to count different molecules to be detected.

[0015] Preferably, the material of the microspheres is one or more of polystyrene, polymethacrylate, silicon dioxide and polyacrylamide; the diameter of the microspheres is 0.1-1000 μm.

[0016] Preferably, the surface of the microsphere is modified by a modifying group, and the modifying group includes one or more of a carboxyl group, an epoxy group, an alkynyl group, an N-hydroxysuccinimide group, an aldehyde group and a streptavidin group.

[0017] Further preferably, the nucleic acid molecule modified on the surface of the microsphere is connected to the modification group on the surface of the microsphere via a specific group, and the specific group includes one or more of an amino group, a thiol group and an epoxy group.

[0018] Preferably, the barcode probe is a single probe.

[0019] The present invention also discloses a method for preparing the above-mentioned barcode repeatable microspheres, comprising the following steps:

[0020] 1) The barcode probe is coupled to the microsphere through a chemical reaction;

[0021] 2) hybridizing the universal primer probe to the barcode probe, and obtaining a capture probe with a cell barcode through polymerization and extension;

[0022] 3) By performing SDA reaction with the Nick region on the barcode probe, a capture probe containing a molecular barcode region, a cell barcode region and a capture region is released, and the molecule to be detected is captured by the capture probe. After the SDA reaction is completed, a capture probe with a cell barcode is obtained, thereby realizing the reuse of the barcode microspheres.

[0023] Preferably, the method of releasing the capture probe comprises SDA, photocleavage or user enzyme cleavage.

[0024] The present invention also discloses the application of the above-mentioned barcoded repeatable microspheres in high-throughput single-cell omics analysis, including:

[0025] The cell suspension is mixed with the barcoded repeatable microspheres, the cells and microspheres are paired, the capture probe is released through the SDA reaction to capture the target to be detected, and the cDNA sequence is obtained through reverse transcription and template conversion, amplified and a sequencing library is constructed to complete the operation of detecting the transcriptome in high-throughput single cells. At the same time, the microspheres can be reused.

[0026] Preferably, the number of cells in the cell suspension and the number of barcoded reproducible microspheres are mixed in a 1:1 ratio.

[0027] Preferably, the reverse transcription treatment temperature is 37-56°C and the time is 1-24h.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The barcode repeatable microsphere disclosed in the present invention includes a microsphere, a universal primer probe and a barcode probe, wherein the barcode probe includes a Nick region, a capture region, a cell barcode region and a molecular barcode region. The barcode repeatable microsphere is based on the SDA reaction to produce repeatable barcode microspheres for high-throughput single-cell omics analysis. Through DNA probe hybridization, polymerization extension, polymer chain displacement and other operations, the barcode microspheres can be used repeatedly, and high-throughput single-cell intracellular gene information detection can be achieved. The present invention is low-cost, simple to operate and has a low threshold. It can be achieved with the help of a microplate, without the need to build an additional microfluidic device, which greatly reduces time and reagent costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of barcoded reproducible microspheres for high-throughput single-cell transcriptome sequencing;

[0031] Figure 2 Schematic diagram of the principle of verifying the repeatability of barcoded reproducible microspheres through standard chains;

[0032] Figure 3 This is a schematic diagram of the structure of a cell barcode library in an embodiment of the present invention;

[0033] Figure 4 This is a fluorescence characterization diagram of the nucleic acid probe modified microspheres;

[0034] Figure 5 Agarose gel electrophoresis diagram for standard strand verification of reproducibility;

[0035] Figure 6Grayscale value diagram of the repeatable situation based on cell verification. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0037] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0038] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0039] The present invention provides a barcode repeatable microsphere, comprising microspheres, universal primer probes and barcode probes. Among them, the universal primer probe is used for PCR amplification; the barcode probe has four parts: Nick region, capture region, cell barcode region and molecular barcode region, wherein the Nick region is used for SDA reaction; the capture region allows the capture probe to be free to capture the detection molecule, which is used for high-throughput single-cell omics analysis, and realizes the repeated use of barcode microspheres; the cell barcode region is used for cell determination; the molecular barcode region is different on each microsphere, and is used to count different molecules to be detected.

[0040] The "Nick area for SDA reaction" described above in the present invention means that when preparing the barcode probe, a specific "Nick area" needs to be introduced on the probe. This area can be a notch or a breakpoint for the subsequent SDA reaction. For example, a notch can be created on the probe through a specific enzymatic reaction, and then the SDA reaction can be used to amplify the nucleic acid. Application of SDA reaction: During the detection process of the barcode probe, if the target biological molecule binds to the probe, the "Nick area" on the probe can be amplified through the SDA reaction. In this way, even if the concentration of the target biological molecule is very low, the signal can be amplified through the SDA reaction, thereby improving the sensitivity of the detection.

[0041] The "capture zone" described above in the present invention is a specific sequence on the barcode probe, which is usually highly complementary to the target molecule, so that specific binding can be achieved. This complementarity can be base complementary pairing (such as complementary pairing between DNA and DNA, RNA and RNA). The design of the capture zone enables the barcode probe to specifically recognize and bind to the target molecule. Once the capture zone binds to the target molecule, the barcode probe can capture the target molecule from a complex biological sample. This makes it possible for subsequent molecular identification, quantitative analysis, etc.

[0042] The "cell barcode region" described above in the present invention refers to a specific region of a cell that is marked by introducing specific markers (such as DNA sequences, fluorescent dyes, radioisotopes, etc.). These markers have unique barcode characteristics and can uniquely identify each cell. On the barcode probe, the cell barcode region is the part that carries these unique barcode information.

[0043] The "molecular barcode region" described above in the present invention refers to a specific sequence on the barcode probe, which serves as a unique molecular identifier (UMI) and is used to assign a unique label to each molecule to be detected, thereby accurately distinguishing and counting each molecule, and achieving high-sensitivity and high-specificity detection even in complex biological samples.

[0044] The present invention also discloses a method for preparing the above-mentioned barcode repeatable microspheres, comprising the following steps:

[0045] 1) The barcode probe is coupled to the microsphere through a chemical reaction;

[0046] 2) hybridizing the universal primer probe to the barcode probe, and obtaining a capture probe with a cell barcode through polymerization and extension;

[0047] 3) By performing SDA reaction with the Nick region on the barcode probe, a capture probe containing a molecular barcode region, a cell barcode region and a capture region is released, and the molecule to be detected is captured by the capture probe. After the SDA reaction is completed, a capture probe with a cell barcode is obtained, thereby realizing the reuse of the barcode microspheres.

[0048] Among them, the SDA reaction releases the nucleic acid probe, which contains a molecular barcode, a cell barcode and a capture area. This step will also polymerize a new nucleic acid probe on the microsphere. This nucleic acid probe is the same as the one released, and it is precisely because of this that the microspheres can be reused.

[0049] like Figure 1 As shown, the present invention provides a method for designing the above-mentioned barcode repeatable microspheres, and the specific operation steps are as follows:

[0050] 1. Design barcoded reproducible microspheres to identify probe sequences of mRNA in tissue cells

[0051] The first group of barcode probes (i.e., probe 1) for microsphere modification is designed. Probe 1 is 20 to 100 bases. Among them, 15 bases are used for SDA reaction, 29 bases are complementary regions of universal primer sequences, 8 bases are the first group of cell barcode regions, and 15 bases are used to connect the second group of barcode probes, which are composed of four parts in total. The 5' end of probe 1 is phosphate-modified, and the 3' end is amino-modified. The amino group is used to bind to carboxyl microspheres. The microsphere modifications include but are not limited to carboxyl and aldehyde groups;

[0052] The second set of barcode probes (i.e., probe 2) for microsphere modification was designed. Probe 2 consists of four parts, starting from 5', which are 30 bases in sequence to form an oligo-dA region, 10 bases for UMI molecular counting, 8 bases for the second set of cell barcodes, and 15 bases for connecting to the first set of barcode probes;

[0053] A probe sequence (ie, probe 3) is designed to connect the first and second groups of barcode probes. Probe 3 consists of two parts: 15 bases for connecting the first group of barcode probes and 15 bases for connecting the second group of barcode probes.

[0054] Design a universal primer probe Read1 sequence (i.e., probe 4) for PCR amplification (PCR handle, PH). Probe 4 consists of two parts: 29 bases of the universal primer probe Read1 sequence for PCR amplification and 15 bases for SDA reaction;

[0055] Design a polyA probe sequence (i.e., probe 5) for standard chain verification. Probe 5 consists of two parts: 20 bases of polyA at the 3' end and 15 bases of universal primer probe Read2 sequence for PCR amplification at the 5' end;

[0056] A probe sequence (ie, probe 6) was designed for template switching. Probe 6 consisted of two parts: a Read2 sequence for PCR amplification at the 5' end and an LNA modification at the 3' end.

[0057] 2. Preparation of barcoded reproducible microspheres

[0058] like Figure 1 As shown, probe 1 with a final concentration of 10 μM is incubated and combined with the functionalized microspheres, probe 2 and probe 3 are subjected to gradient annealing hybridization, and incubated with the microspheres modified with probe 1 for later use.

[0059] like Figure 1 As shown, capture probes with cell barcodes were generated by SDA reaction for standard strand and tissue section verification.

[0060] like Figure 2 As shown, the barcode microspheres that have completed functional modification are used for standard chain verification. In the presence of probe 5, the capture probes produced by the SDA reaction will bind to it and stably hybridize, and the above capture probes will dissociate from the barcode microspheres, making them reusable. The dissociated probes are amplified by PCR, and the size of the electrophoresis bands is verified by agarose gel electrophoresis. After completing the first round of verification, continue to repeat the above operations to complete the verification of the repeatability of the SDA barcode microspheres.

[0061] 3. Application of barcoded reproducible microspheres for single-cell omics analysis

[0062] like Figure 1 As shown in the figure, the barcode microspheres with functional modification are used for cell sample verification. First, the SDA reaction produces the capture probe. Then, after the cells are lysed, the capture probe will bind to the mRNA molecule and perform the reverse transcription process to encode the genetic information in the cell. The complete capture probe will dissociate from the barcode microsphere, making it reusable. The probe with cDNA obtained by reverse transcription is amplified by PCR, as shown in the figure. Figure 3 As shown, library construction and sequencing are used to analyze genetic information within cells.

[0063] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0064] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples are conventional commercial products unless otherwise specified, and their specifications are conventional specifications in the art. In the specification of the present invention and the following examples, unless otherwise specified, "%" represents mass percentage and ratio represents mass ratio.

[0065] 1. Design of nucleic acid probe molecules and their simulation:

[0066] In order to realize the use of barcoded reproducible microspheres for cytomic analysis, corresponding nucleic acid probes were designed.

[0067] In the process of designing probe 1, based on the characteristics of rapid coupling between carboxyl and amino groups, one end of the amino group was modified to bind to the carboxyl microsphere; 16 bases were designed as the cell barcode region for cell identification; in order to reduce steric hindrance, a spacer was designed between the microsphere and the functional region; and in order to achieve SDA reaction, a Nick site was designed. The sequence of probe 1 is shown in SEQ ID NO. 1 to 6 in Table 1, indicating that probe 1 can be any one of 1-1, 1-2, 1-3, 1-4, 1-5 and 1-6 in Table 1. .

[0068] In the process of designing probe 2, according to the principle of base complementary pairing, 30 polyA regions are designed to generate poly T. The sequence of probe 2 is shown in SEQ ID NO. 7 to 12 in Table 2, indicating that probe 2 can be any one of 2-1, 2-2, 2-3, 2-4, 2-5 and 2-6 in Table 1.

[0069] In the process of designing probe 3, 15 bases were designed to stably connect probe 1 and probe 2. The sequence of probe 3 is shown in SEQ ID NO. 13 in Table 1.

[0070] In the process of designing probe 4, the read1 region is designed according to the primers required in the PCR amplification process; the hybridization region is designed for the hybridization Nick region. The sequence of probe 4 is shown in SEQ ID NO. 14 in Table 1.

[0071] In the process of designing probe 5, the read2 region is designed according to the primers required in the PCR amplification process, and the polyA sequence is designed for hybridization probe 2. The sequence of probe 4 is shown in SEQ ID NO. 15 in Table 1.

[0072] In the process of designing probe 6, the read2 region is designed according to the primers required in the PCR amplification process, and the LNA sequence is designed at the 3' end for hybridization of the reverse transcription product. The sequence of probe 6 is shown in SEQ ID NO. 16 in Table 1.

[0073] Table 1 Part of the first group of barcode probe sequences

[0074]

[0075]

[0076] Table 2 Partial second group barcode probe sequences

[0077]

[0078] Table 2 Some related sequences of the examples

[0079]

[0080] The above probes 1, 2, 3, 4, 5 and 6 were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and used in the following experiments. Note that probes 1 and 2 can be designed in various ways as shown in Tables 1 and 2 above.

[0081] 2. Preparation of barcode microspheres modified with probe 1, probe 2 and probe 3:

[0082] Combine probe 1 at a concentration of 10 μM with the carboxyl glass slide and incubate with shaking at room temperature overnight. Use 1×PBST solution (containing 1% BSA) to wash the surface of the microspheres to remove non-specifically adsorbed DNA. Perform gradient annealing on probes 2 and 3: cool from 95°C to 20°C at a rate of 0.1°C per second. Then hybridize with probe 1 at 37°C for 30 minutes. Wash the surface of the microspheres with 1×PBST solution (containing 1% BSA) to remove unreacted and non-specifically adsorbed DNA. Use DNA ligase to connect probe 1 and probe 2 (only some probe sequences are listed in Tables 1 and 2), and use KOH solution to remove probe 3 for reusable SDA barcode microspheres. Figure 4 As shown, fluorescence microscopy characterized the barcode probe connection status of the two groups of cells.

[0083] 3. Barcode reproducible microspheres for standard chain verification

[0084] The probe 4 at a concentration of 1 μM is reacted with the barcode microspheres prepared in step 2. After reacting at 37°C for 2 hours, the surface of the microspheres is washed with 1×PBST solution (containing 1% BSA) to remove unreacted and non-specifically adsorbed DNA. DNA polymerase is incubated with the microspheres at 37°C for 2 hours to perform SDA reaction to obtain capture probes with cell barcodes. The capture probes are polymerized and extended with probe 5 at a concentration of 1 μM, and the above products are amplified by PCR. The barcode microspheres continue to undergo polymerization extension and SDA reaction, and this is repeated so that the barcode microspheres can be reused. Figure 5As shown, three rounds of reproducibility experiments were performed, and PCR real-time curves and agarose gel electrophoresis were used to characterize the reproducibility.

[0085] 4. Barcoded reproducible microspheres for cell sample verification

[0086] Select tumor cells MCF-7, prepare a cell suspension of appropriate concentration, and perform cell activity analysis. The analysis effect is best when the viable cell rate is >90%. After the cell suspension and barcode microspheres are evenly mixed, add cell lysis solution and SDA reaction solution, shake and react at 37°C for 30 minutes to perform cell lysis and mRNA capture. Then take the supernatant, add reverse transcription reagent for reverse transcription and template conversion, react at 42°C for 6 hours, and reverse transcribe mRNA into cDNA. After completion, the solution contains cDNA sequences with barcodes, which are amplified by PCR. Repeat this process, such as Figure 6 As shown, three rounds of reproducibility validation were performed, and PCR real-time curves and agarose gel electrophoresis were used to characterize its reproducibility.

[0087] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A barcode repeatable microsphere, characterized in that: Includes microspheres, universal primer probes, and barcode probes; The barcode probe is coupled to the microsphere, the universal primer probe can hybridize with the barcode probe, and the universal primer probe is used for PCR amplification; The barcode probe includes a Nick region, a capture region, a cell barcode region and a molecular barcode region, wherein: Nick region, used for SDA reaction; A capture zone, used to capture the molecules to be detected; Cell barcode region, used for cell identification; The molecular barcode region is used to count different molecules to be detected.

2. The barcode repeatable microsphere according to claim 1, characterized in that: The material of the microspheres is selected from one or more of polystyrene, polymethacrylate, silicon dioxide and polyacrylamide; the diameter of the microspheres is 0.1-1000 μm.

3. The barcode repeatable microsphere according to claim 1, characterized in that: The surface of the microsphere is modified by a modification group, and the modification group includes one or more of a carboxyl group, an epoxy group, an alkynyl group, an N-hydroxysuccinimide group, an aldehyde group and a streptavidin group.

4. The barcode repeatable microsphere according to claim 3, characterized in that: The nucleic acid molecules on the surface of the microspheres are connected to the modified groups on the surface of the microspheres through specific groups, and the specific groups include one or more of amino groups, thiol groups and epoxy groups.

5. The barcode repeatable microsphere according to claim 1, characterized in that: The barcode probe is a single probe.

6. The method for preparing the barcode repeatable microspheres according to any one of claims 1 to 5, characterized in that: The following steps are involved: 1) The barcode probe is coupled to the microsphere through a chemical reaction; 2) hybridizing the universal primer probe to the barcode probe, and obtaining a capture probe with a cell barcode through polymerization and extension; 3) Through SDA reaction with the Nick region on the barcode probe, a capture probe containing a molecular barcode region, a cell barcode region and a capture region is released, and the molecule to be detected is captured by the capture probe; after the SDA reaction is completed, a capture probe with a cell barcode is obtained, thereby realizing the reuse of the barcode microspheres.

7. The method for preparing barcode repeatable microspheres according to claim 6, characterized in that: Methods for releasing capture probes include SDA, photocleavage, or user enzyme cleavage.

8. Use of the barcoded repeatable microspheres according to any one of claims 1 to 5 in high-throughput single-cell omics analysis, characterized in that: include: The cell suspension is mixed with the barcoded repeatable microspheres, the cells and microspheres are paired, the capture probe is released through the SDA reaction to capture the target to be detected, and the cDNA sequence is obtained through reverse transcription and template conversion, amplified and a sequencing library is constructed to complete the operation of detecting the transcriptome in high-throughput single cells. At the same time, the microspheres can be reused.

9. The use according to claim 8, characterized in that: The number of cells in the cell suspension and the number of barcoded reproducible microspheres are mixed in a 1:1 ratio.

10. The use according to claim 8, characterized in that: The reverse transcription treatment temperature is 37-56°C and the time is 1-24h.

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