A barcode repeatable microsphere, its preparation method and application

Abstract

The application discloses a bar code repeatable microsphere, a preparation method and application thereof, and relates to the fields of biomedical detection and high-throughput single cell sequencing.The bar code repeatable microsphere comprises a microsphere, a universal primer probe and a bar code probe, and the bar code probe comprises a Nick region, a capture region, a cell bar code region and a molecular bar code region.The method can realize preparation of the bar code microsphere and repetition of the bar code at a very low cost, is simple to operate, and is widely applied, and can be used for detection in the aspects of genomes, transcriptomes, proteomes, epigenomes and metabolomes.

Description

Technical Field

[0001] This invention relates to the fields of biomedical detection and high-throughput single-cell sequencing, and particularly to a method for preparing barcode repeatable microspheres and their applications. Background Technology

[0002] Biomedical testing is a science that applies biomedical principles and methods to detect, measure, and analyze organisms and their components using specific technical means. It involves multiple principles, such as optics, electrochemistry, immunology, and biology, and plays a crucial role in medical diagnosis, treatment monitoring, disease prevention, drug development, and biomedical research. Biomedical testing encompasses several aspects, including gene detection, protein detection, metabolite detection, cell analysis, and bioimaging. Among these, Single Cell Analysis (SCA) is a method for in-depth study of individual cells, aiming to reveal information about single cells at the genomic, transcriptomic, epigenomic, and proteomic levels. Through SCA, gene variations and changes in gene expression within individual cells can be detected, aiding in early disease diagnosis. In oncology, SCA can identify tumor cell subpopulations with different gene expression patterns, providing a basis for personalized treatment. In basic medical research, SCA can be used to study biological processes such as stem cell differentiation and apoptosis, providing new insights for regenerative medicine and disease treatment. Therefore, single-cell analysis plays a crucial role in biomedical testing, helping to improve the accuracy of disease diagnosis, enable personalized medicine, and accelerate drug development. With continuous technological advancements and expanding applications, single-cell analysis will play an even more vital role in the future of biomedicine.

[0003] Barcoded microspheres play a crucial role in single-cell analysis, particularly in high-throughput single-cell sequencing and analysis. A barcoded microsphere is a tiny sphere with a unique barcode identifier. The barcode probes on the microsphere typically include the following functional regions: universal primers, cell barcodes, molecular barcodes, and capture regions. The use of barcoded microspheres enables high-throughput sequencing technology to be applied at the single-cell level. Each cell is assigned a unique barcode, allowing for accurate differentiation of sequencing data from different cells even in mixed samples, significantly improving the efficiency and accuracy of single-cell sequencing.

[0004] Currently, high-throughput single-cell sequencing barcode microspheres mainly include the following types: 1) Macosko-2011-10(V+) from ChemGenes, a common commercial barcode microsphere typically used in high-throughput single-cell sequencing technology. It is made of resin material and its surface is modified with oligonucleotides for capturing and labeling mRNA poly-dT sequences, cell barcode sequences to distinguish different cells, molecular barcode sequences for molecular quantification, and universal adapter sequences for downstream next-generation sequencing; 2) Gel Beads-in-emulsions (GEMs) from 10x Genomics, which are gel microspheres modified with nucleic acid probes. These nucleic acid probes also include functional regions such as poly-dT, cell barcodes, molecular barcodes, and universal adapters. During the use of GEMs, cells are restrictedly diluted to ensure that most GEMs do not contain cells, while GEMs containing cells mostly contain only single cells. Subsequently, within the GEMs, the cells are lysed, the gel beads dissolve, and a large number of barcode-laden nucleic acid probes are released. These probes capture mRNA with a PolyA tail and reverse transcribe it to generate the first strand of cDNA with 10x barcode and UMI information.

[0005] However, in the aforementioned Macosko-2011-10(V+) microspheres, the nucleic acid probes with barcode functional regions participate in cDNA synthesis during reverse transcription, leading to the consumption of the barcode probes and rendering the microspheres unusable. Similarly, 10x Genomics' gel microspheres with barcode probes dissolve and are consumed during use, also making them unusable. Furthermore, the preparation of barcode microspheres requires specialized equipment and complex processes, and high-throughput single-cell sequencing demands extremely high quality from both the microspheres and the barcodes; otherwise, the final sequencing results will be affected. This is why, to date, only Macosko-2011-10(V+) is a commercially available barcode microsphere. On the one hand, this results in the high cost of current high-throughput single-cell sequencing microspheres; on the other hand, the limited variety of commercially available barcode microspheres fails to meet the current requirements for multi-omics and multi-target detection, significantly restricting the flexibility of sequencing technology applications. Therefore, developing a simple and low-cost barcode-reproducible microsphere is of great significance for high-throughput single-cell analysis. Summary of the Invention

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

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] This 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 microspheres, and the universal primer probe can hybridize with the barcode probe. The universal primer probe is used for PCR amplification; the universal primer probe contains a Nick region and a universal primer sequence region.

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

[0011] The Nick hybridization region can be complementary to the Nick region for the SDA reaction;

[0012] The capture region is used to capture the molecules to be detected.

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

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

[0015] Preferably, the microspheres are made of one or more of polystyrene, polymethacrylate, silica, and polyacrylamide; the diameter of the microspheres is 0.1-1000 μm.

[0016] Preferably, the surface of the microspheres is modified by modifying groups, which include one or more of carboxyl, epoxy, alkynyl, N-hydroxysuccinimide, aldehyde and streptavidin groups.

[0017] More preferably, the nucleic acid molecules modified on the surface of the microspheres are linked to the modifying groups on the surface of the microspheres through specific groups, wherein the specific groups include one or more of amino, thiol and epoxy groups.

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

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

[0020] 1) Coupling the barcode probe to the microspheres via a chemical reaction;

[0021] 2) Hybridize universal primer probes to barcode probes, and obtain capture probes with cell barcodes through polymerization and extension;

[0022] 3) An SDA reaction is performed using the Nick region on a universal primer probe to release a capture probe containing a molecular barcode region, a cell barcode region, and a capture region. The target molecule is captured by this capture probe. After the SDA reaction is complete, a capture probe with a cell barcode is obtained, enabling the reuse of barcode microspheres.

[0023] Preferably, the method for releasing the capture probe includes SDA, photodigestion, or user enzyme digestion.

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

[0025] The cell suspension is mixed with the barcode repeatable microspheres, the cells and microspheres are paired, and the capture probe is released through the SDA reaction to capture the target analyte. The cDNA sequence is obtained through reverse transcription and template conversion, amplified and constructed into a sequencing library, thus completing 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 is mixed with the number of barcode repeatable microspheres at a 1:1 ratio.

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

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

[0029] The barcode-reproducible microspheres disclosed in this invention comprise microspheres, universal primer probes, and barcode probes. The barcode probes include a Nick hybridization region, a capture region, a cellular barcode region, and a molecular barcode region. These barcode-reproducible microspheres are based on an SDA reaction, generating reproducible barcode microspheres for high-throughput single-cell omics analysis. Through DNA probe hybridization, polymerization extension, and polymer chain replacement, the barcode microspheres can be reused repeatedly, enabling high-throughput detection of intracellular gene information in single cells. This invention is low-cost, simple to operate, and has a low barrier to entry. It can be implemented using only microplates, without the need for additional microfluidic devices, significantly reducing time and reagent costs. Attached Figure Description

[0030] Figure 1 Schematic diagram of barcode repeatable microspheres used for high-throughput single-cell transcriptome sequencing;

[0031] Figure 2 A schematic diagram illustrating the principle of verifying the repeatability of barcode-repeatable microspheres using a standard chain;

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

[0033] Figure 4 Fluorescence characterization of microspheres modified with nucleic acid probes;

[0034] Figure 5 Agarose gel electrophoresis image for verifying reproducibility of the standard strand;

[0035] Figure 6 This is a grayscale image showing the reproducibility of cell-based validation. Detailed Implementation

[0036] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0038] The present invention will now be described in further detail with reference to the accompanying drawings:

[0039] This invention provides a barcode-repeatable microsphere comprising three parts: a microsphere, a universal primer probe, and a barcode probe. The universal primer probe is used for PCR amplification and contains a Nick region and a universal primer sequence region. The barcode probe has four parts: a Nick hybridization region, a capture region, a cell barcode region, and a molecular barcode region. The Nick region is complementary to the Nick hybridization region and is used for SDA reactions. The capture region allows the capture probe to capture and detect molecules, enabling high-throughput single-cell omics analysis and allowing for the reuse of the barcode microsphere. The cell barcode region is used for cell identification. The molecular barcode region is different on each microsphere and is used to count different detectable molecules.

[0040] The "Nick region for SDA reaction" described above in this invention refers to the introduction of a specific "Nick region" onto the probe during barcode probe preparation. This region can be a notch or breakpoint for subsequent SDA reactions. For example, a notch can be created on the probe through a specific enzymatic reaction, followed by nucleic acid amplification using the SDA reaction. Applications of the SDA reaction: If the target biomolecule binds to the probe, the "Nick region" on the probe can be amplified using the SDA reaction. Thus, even at low concentrations of the target biomolecule, signal amplification can be achieved through the SDA reaction, thereby improving detection sensitivity.

[0041] The "capture region" described above in this invention is a specific sequence on a barcode probe that typically exhibits high complementarity with the target molecule, thereby enabling specific binding. This complementarity can be base pairing (such as complementary pairing between DNA and DNA, or RNA and RNA). The design of the capture region allows the barcode probe to specifically recognize and bind to the target molecule. Once the capture region binds to the target molecule, the barcode probe can capture the target molecule from complex biological samples. This enables subsequent molecular identification, quantitative analysis, and other processes.

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

[0043] The “molecular barcode region” described above in this invention refers to a specific sequence on the barcode probe, which serves as a unique molecular identifier (UMI) to assign a unique tag 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] This invention also discloses a method for preparing the above-mentioned barcode repeatable microspheres, comprising the following steps:

[0045] 1) Coupling the barcode probe to the microspheres via a chemical reaction;

[0046] 2) Hybridize universal primer probes to barcode probes, and obtain capture probes with cell barcodes through polymerization and extension;

[0047] 3) An SDA reaction is performed using the Nick region on a universal primer probe to release a capture probe containing a molecular barcode region, a cell barcode region, and a capture region. The target molecule is captured by this capture probe. After the SDA reaction is complete, a capture probe with a cell barcode is obtained, enabling the reuse of barcode microspheres.

[0048] In this process, the SDA reaction releases a nucleic acid probe containing molecular barcodes, cell barcodes, and a capture region. This step also polymerizes a new nucleic acid probe on the microsphere, which is the same as the one that was released. This is why the microspheres can be reused.

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

[0050] 1. Design barcode-repeatable microspheres for identifying probe sequences of mRNA within tissue cells.

[0051] The first set of barcode probes (i.e., probe 1) for microsphere modification is designed, consisting of 20-100 bases. Of these, 15 bases are used for the SDA reaction, 29 bases are the complementary region of the universal primer sequence, 8 bases are the first set of cell barcode regions, and 15 bases are used to connect to the second set of barcode probes, making a total of four parts. Probe 1 is modified with a phosphate group at its 5' end and an amino group at its 3' end. The amino group is used to bind carboxyl microspheres. The microsphere modification includes, but is not limited to, carboxyl and aldehyde groups.

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

[0053] The probe sequence (i.e., probe 3) is designed to connect the first and second sets of barcode probes. Probe 3 consists of two parts: 15 bases for connecting the first set of barcode probes and 15 bases for connecting the second set of barcode probes.

[0054] The universal primer probe Read1 sequence (i.e. probe 4) for PCR amplification (PCR handle, PH) is designed. Probe 4 consists of two parts: a 29-base universal primer probe Read1 sequence for PCR amplification and a 15-base sequence for SDA reaction.

[0055] The polyA probe sequence (i.e. probe 5) designed for standard chain verification consists of two parts: a 20-base polyA sequence at the 3' end and a 15-base universal primer probe Read2 sequence at the 5' end for PCR amplification.

[0056] The probe sequence designed for template conversion (i.e., probe 6) consists of two parts: a Read2 sequence at the 5' end for PCR amplification and an LNA modification at the 3' end.

[0057] 2. Preparation of barcode repeatable microspheres

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

[0059] like Figure 1 As shown, a capture probe with a cell barcode is generated via an SDA reaction for verification on standard strands and tissue sections.

[0060] like Figure 2 As shown, the functionalized barcode microspheres were used for standard chain validation. In the presence of probe 5, the capture probe generated by the SDA reaction binds to and stably hybridizes with it, and the capture probe dissociates from the barcode microsphere, allowing for reusability. The dissociated probe was subjected to PCR amplification, and the size of the electrophoretic bands was verified by agarose gel electrophoresis. After completing the first round of validation, the above operation was repeated to verify the reproducibility of the SDA barcode microspheres.

[0061] 3. Application of single-cell omics analysis in barcode repeatable microspheres

[0062] like Figure 1 As shown, functionalized barcode microspheres are used for cell sample validation. First, an SDA reaction generates a capture probe. Subsequently, after cell lysis, the capture probe binds to mRNA molecules, and during reverse transcription, it encodes intracellular genetic information. The intact capture probe dissociates from the barcode microsphere, allowing for reuse. The probe containing cDNA obtained from reverse transcription is then subjected to PCR amplification, as shown... Figure 3 As shown, library construction and sequencing are used to analyze intracellular genetic information.

[0063] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0064] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" represents a percentage by mass, and "ratio" represents a mass ratio.

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

[0066] To enable barcode-repeatable microspheres to be used for cell omics analysis, corresponding nucleic acid probes were designed.

[0067] In designing probe 1, based on the rapid coupling properties of carboxyl and amino groups, an amino group was modified at one end to bind to the carboxyl microsphere. A 16-base region was designed as a cell barcode region for cell identification. To reduce steric hindrance, a spacer region was designed between the microsphere and the functional region. Simultaneously, a Nick hybridization site was designed to enable the SDA reaction. The sequence of probe 1 is shown in Table 1 as SEQ ID NO. 1~6, 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 design of probe 2, 30 polyA regions were designed to generate polyT regions based on the base complementary pairing principle. The sequence of probe 2 is shown in SEQ ID NO. 7~12 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 design of 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 designing probe 4, the read1 region was designed based on the primers required for PCR amplification. The sequence of probe 4 is shown in SEQ ID NO.14 in Table 1.

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

[0072] In designing probe 6, the read2 region was designed based on the primers required for PCR amplification, serving as the reverse transcription product of the hybridization, and the 3' end was designed with an LNA sequence. The sequence of probe 6 is shown in SEQ ID NO.16 in Table 1.

[0073] Table 1. Partial Group 1 Barcode Probe Sequences

[0074]

[0075] Table 2 Partial Group 2 Barcode Probe Sequences

[0076]

[0077] Table 3. Related sequences in some examples

[0078]

[0079] 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, as shown in Tables 1 and 2 above, can be designed in various ways.

[0080] 2. Preparation of barcode microspheres modified with probe 1, probe 2, and probe 3:

[0081] Probe 1 at a concentration of 10 μM was bound to a carboxyl glass slide and incubated overnight at room temperature with shaking. The microsphere surface was washed with 1×PBST solution (containing 1% BSA) to remove non-specifically adsorbed DNA. Probes 2 and 3 were subjected to gradient annealing: decreasing from 95°C to 20°C at a rate of 0.1°C per second. They were then hybridized with probe 1 at 37°C for 30 min. The microsphere surface was washed with 1×PBST solution (containing 1% BSA) to remove unreacted and non-specifically adsorbed DNA. Probes 1 and 2 were ligated using DNA ligase (only some probe sequences are listed in Tables 1 and 2), and probe 3 was removed with KOH solution for reusability of SDA barcode microspheres. Figure 4 As shown, fluorescence microscopy characterizes the connection between the two sets of cell barcode probes.

[0082] 3. Barcode repeatable microspheres for standard chain verification

[0083] The barcode microspheres prepared in step 2 were reacted with 1 μM probe 4 at 37°C for 2 h. The microspheres were then washed with 1×PBST solution (containing 1% BSA) to remove unreacted and non-specifically adsorbed DNA. DNA polymerase was then incubated with the microspheres at 37°C for 2 h to perform an SDA reaction, yielding a capture probe carrying a cell barcode. This capture probe was then polymerized and extended with 1 μM probe 5, and the product was amplified by PCR. The barcode microspheres were repeatedly subjected to polymerization extension and SDA reactions, allowing for reuse. Figure 5 As shown, three rounds of reproducibility experiments were conducted, and the reproducibility was characterized by real-time PCR curves and agarose gel electrophoresis.

[0084] 4. Barcode-repeatable microspheres for cell sample verification

[0085] MCF-7 tumor cells were selected, and a cell suspension of appropriate concentration was prepared for cell viability analysis. The best analytical results were obtained when the viable cell rate was >90%. The cell suspension was mixed thoroughly with barcode microspheres, and then cell lysis buffer and SDA reaction solution were added. The mixture was incubated at 37°C with shaking for 30 min to lyse the cells and capture mRNA. The supernatant was then collected, and reverse transcription reagent was added for reverse transcription and template conversion. The reaction was carried out at 42°C for 6 h, and mRNA was reverse transcribed into cDNA. After the reaction, the solution contained the barcode-encoded cDNA sequence, which was then amplified by PCR. This process was repeated as follows... Figure 6 As shown, three rounds of reproducibility verification were performed, and the reproducibility was characterized by real-time PCR curves and agarose gel electrophoresis.

[0086] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A barcoded microsphere, characterized in that, The microsphere, the barcode probe and the universal primer probe are included. The barcode probe is coupled to the microsphere by chemical reaction. The barcode probe contains a Nick hybridization region, a universal primer sequence complementary region, a capture region, a cell barcode region and a molecular barcode region. The universal primer probe contains a Nick region and a universal primer sequence region, which are used for polymerization, extension and SDA reaction. The Nick hybridization region is used for complementary pairing with the Nick region, the universal primer sequence complementary region is used for complementary pairing with the universal primer sequence region, the capture region is used for generating a capture sequence after hybridization of the barcode probe and the universal primer probe through polymerization and extension, the cell barcode region is used for cell determination, and the molecular barcode region is used for counting the molecules to be detected. The universal primer probe generates a capture probe containing a molecular barcode sequence, a cell barcode sequence and a capture sequence after hybridization to the barcode probe through polymerization and extension; the Nick region is used for specific cutting by nicking enzyme to generate a gap and perform SDA reaction to release the capture probe, which is used for capturing the molecules to be detected through the capture sequence.

2. The barcoded microsphere of claim 1, wherein, The material of the microsphere is selected from one or more of polystyrene, polymethacrylate, silica and polyacrylamide; the diameter of the microsphere is 0.1-1000 μm.

3. The barcodable microsphere of claim 1, wherein, The surface of the microsphere is modified by a modification group, and the modification group includes one or more of carboxyl, epoxy, alkyne, N-hydroxysuccinimide, aldehyde and streptavidin groups.

4. The barcoded microsphere of claim 3, wherein, The nucleic acid molecule on the barcode probe is connected to the modification group on the surface of the microsphere through a specific group, and the specific group includes one or more of amino, thiol and epoxy.

5. The method of claim 1-4, wherein the method of preparing and reusing the barcoded microsphere is characterized in that, The method includes the following steps: 1) coupling the barcode probe to the microsphere by chemical reaction; 2) hybridizing the universal primer probe to the barcode probe to obtain a capture probe containing a molecular barcode sequence, a cell barcode sequence and a capture sequence through polymerization and extension reaction; 3) releasing the capture probe by specific cutting of the Nick region by nicking enzyme and performing SDA reaction, and capturing the molecules to be detected by using the capture probe; 4) performing SDA reaction again in step 3) to obtain the capture probe again, and realizing repeated use of the barcode repeatable microsphere.

6. Use of the barcoded microsphere according to any one of claims 1 to 4 in high-throughput single-cell omics analysis, characterized in that, The method includes: mixing the cell suspension with the barcode repeatable microsphere to make the cells and the microspheres pair, and then releasing the capture probe by specific cutting of the nicking enzyme and SDA reaction to capture the mRNA in the cells, at this time the barcode repeatable microsphere can be reused; then obtaining cDNA sequence through reverse transcription, amplifying and constructing sequencing library, and finally performing sequencing and high-throughput single cell omics analysis.

7. Use according to claim 6, characterized in that, The number of cells in the cell suspension and the number of barcode repeatable microspheres are mixed at a ratio of 1:

1.

8. Use according to claim 6, characterized in that The reverse transcription processing temperature is 37-56℃, and the time is 1-24h.

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