A bar code reproducible microsphere, a preparation method, a regeneration method and application thereof

Abstract

The application discloses a bar code regenerable microsphere, a preparation method, a regeneration method and application thereof, and relates to the fields of biomedical detection and high-throughput single cell sequencing.The bar code regenerable microsphere disclosed by the application is composed of seven parts, i.e., a microsphere, a universal primer, a cell bar code, a hybridization region, a strand displacement region, a molecular bar code and a capture region.Based on the bar code regenerable microsphere, high-throughput single cell analysis is carried out, the bar code microsphere can be repeatedly used through DNA probe hybridization, polymerization extension and polymer chain displacement, and the detection of gene information in high-throughput single cells can be realized.The application has the advantages of low cost, simple operation and low threshold.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical detection and high-throughput single-cell sequencing, and particularly relates to a barcode reproducible microsphere, a preparation method and application thereof. BACKGROUND

[0002] Biomedical detection plays a very significant role in gene detection, drug screening, disease diagnosis, environmental monitoring, etc., and therefore the development of efficient, simple and low-cost detection methods has become the focus of many researchers. The latest progress in single-cell analysis shows that even cells of the same type exhibit significant heterogeneity, including differences in fate, morphology and differential expression of biomolecules. Therefore, analyzing cells at the single-cell level helps us have a more comprehensive understanding of life processes, and high-throughput single-cell detection technology has become one of the most important detection methods in biomedical detection due to its high precision, multi-dimensionality and high-throughput characteristics.

[0003] Barcode microspheres are important tools in high-throughput single-cell detection technology. The barcode probes on the microspheres usually include the following functional regions: universal primers, cell barcodes, molecular barcodes and capture regions. The universal primer region is used as a template for amplification during library construction, and nucleic acid chains with barcodes and target sequences are amplified for library construction; the cell barcode is usually designed as a specified nucleotide chain, and the cell barcode is unique in a single microsphere, and the cell codes on different microspheres are different, which is used to distinguish different cells; the molecular barcode is usually designed as a completely random nucleotide chain (such as NNNNNNN), a partially degenerate nucleotide chain (such as NNNRNYN), which is used for molecular counting and correcting sequencing errors and PCR errors in the sequencing data; the capture region is usually designed as the complementary sequence of the characteristic sequence of the molecule to be detected, which is used to identify and capture the molecule to be detected.

[0004] Currently, high-throughput single-cell sequencing barcode microspheres mainly include the following: 1) Macosko-2011-10 (V+) provided by ChemGenes Company, which is a commercialized barcode ball and is usually used in high-throughput single-cell sequencing technology. The barcode ball is usually made of resin material, and the surface is modified with oligonucleotides, which include 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 Beads-in-emulsions (GEMs) of 10x Genomics Company, which is a gel microsphere with oligonucleotides, and the oligonucleotides also include poly-dT, cell barcode, molecular barcode and universal adapter. In the use process of GEMs, the cells are restrictedly diluted to ensure that most GEMs do not contain cells, and the GEMs containing cells contain only a single cell. Then in the GEMs, the cells are lysed, the gel beads are dissolved, and a large number of primer sequences are released, which are reverse transcribed with the mRNA with PolyA tail to generate cDNA first strand with 10x Barcode and UMI information.

[0005] However, in the above high-throughput single-cell sequencing barcode microspheres, the oligonucleotide sequences containing barcode probes will be polymerized and extended during reverse transcription, that is, they will participate in the synthesis of new chains and become part of the new chains. Since the barcode probe is integrated into the cDNA chain during reverse transcription, the barcode probe is consumed and cannot be reused. On the one hand, it will increase the cost and may produce a large number of waste barcode probes during sequencing, causing resource waste; on the other hand, the consumption of barcode probes limits the flexibility and scalability of the sequencing technology. The traditional method is very complex and time-consuming in preparing barcodes, and the prepared barcodes (microspheres) cannot be used again. Therefore, it is of great significance to develop a simple and low-cost barcode regenerable microsphere for high-throughput single-cell analysis. SUMMARY

[0006] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a barcode regenerable microsphere and a preparation method and application thereof, so as to solve the technical problems that the existing barcode microspheres are disposable, cannot be reused and the preparation process is relatively complex.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0008] The present application discloses a barcode regenerable microsphere, which comprises a microsphere, a barcode probe, a capture probe, a universal primer probe and a strand displacement probe.

[0009] The barcode probe is coupled to the microsphere;

[0010] The barcode probe is provided with a universal primer hybridization region, a cell barcode region and a capture probe hybridization region; wherein the universal primer hybridization region is used for complementary pairing with the universal primer probe, the cell barcode region is used for cell determination, and the capture probe hybridization region is used for complementary pairing with the capture probe;

[0011] The capture probe is provided with a barcode probe hybridization region, a strand displacement probe hybridization region and a capture region; wherein the barcode probe hybridization region is used for complementary pairing with the capture probe hybridization region on the barcode probe, the strand displacement probe hybridization region is used for complementary pairing with the strand displacement probe, and the capture region is used for capturing mRNA in cells;

[0012] The universal primer probe and the capture probe are used for generating a full-length capture probe containing a universal primer probe sequence, a cell barcode sequence and a capture probe sequence through hybridization to the barcode probe and then polymerization and ligation reactions;

[0013] The strand displacement probe is used for complementary pairing with the strand displacement probe hybridization region on the capture probe and performing a polymerization strand displacement reaction to displace and release the full-length capture probe.

[0014] Preferably, the capture probe is further provided with a molecular barcode region, and the molecular barcode is different on a single microsphere and is used for coding different molecules to be detected.

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

[0016] Preferably, 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 alkyne group, an N-hydroxysuccinimide group, an aldehyde group and streptavidin; and the nucleic acid 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 an amino group, a sulfydryl group and an epoxy group.

[0017] The application further discloses a preparation and regeneration method of the barcode regenerable microsphere.

[0018] 1) coupling the barcode probe to the microsphere through a chemical reaction to obtain a barcode microsphere;

[0019] 2) hybridizing the universal primer probe and the capture probe to the barcode probe, and generating a full-length capture probe containing a universal primer probe sequence, a cell barcode sequence and a capture probe sequence through polymerization and ligation reactions.

[0020] 3) Capture the molecules to be detected by using the barcode microspheres obtained in step 2), and hybridize the strand displacement probe to the strand displacement probe hybridization region on the capture probe and perform a polymerase chain displacement reaction to displace and release the full-length capture probe capturing the molecules to be detected, for library construction;

[0021] 4) The barcode microspheres after step 3) are subjected to the polymerization and ligation of step 2) again to generate full-length capture probes again, so as to regenerate the barcode regenerable microspheres.

[0022] The application further discloses application of the barcode regenerable microspheres in single-cell omics analysis, comprising the following steps:

[0023] The cell suspension is mixed with the barcode regenerable microspheres, cell lysis and mRNA capture are performed, and then reverse transcription is performed to convert mRNA into cDNA; after the polymerase chain displacement reaction, the full-length capture probe capturing the cDNA sequence is displaced and released, and a sequencing library is constructed, at this time, the barcode regenerable microspheres can be regenerated and used; finally, sequencing and single-cell omics analysis are performed.

[0024] Preferably, the number of cells in the cell suspension is mixed with the number of barcode regenerable microspheres at 1:1.

[0025] Preferably, the temperature of the reverse transcription is 37-56℃, and the time is 1-24 h.

[0026] The application further discloses application of the barcode regenerable microspheres in constructing a sequencing library.

[0027] Compared with the prior art, the application has the following beneficial effects:

[0028] The barcode regenerable microspheres disclosed by the application comprise a microsphere, a barcode probe, a capture probe, a universal primer probe and a strand displacement probe, for the first time, molecular barcodes and capture regions are designed on two nucleic acid probes, cell barcodes and functional regions such as molecular barcodes are unified to one nucleic acid probe by polymerization and ligation, and a nucleic acid probe with complete functional regions is obtained. In addition, the method innovatively adds two functional regions of a hybridization region and a strand displacement region to the nucleic acid probe of the barcode microsphere, the cell barcode on the microsphere is retained by polymerase chain displacement, so that the cell barcode probe connected to the microsphere is not consumed when the microsphere is used, thereby realizing the regenerability of the barcode, and greatly reducing the time cost and money cost in preparation of the barcode microsphere. Therefore, the application realizes the detection of high-throughput single-cell gene information by DNA probe hybridization, polymerization and extension, and polymerase chain displacement, so that the barcode microsphere can be repeatedly used. The application has low cost, simple operation and low threshold.

[0029] Further, only need to help micro-hole plate during production, no need to build micro-fluidic device additionally, greatly reduce time, reagent cost. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 For bar code-based regenerable microspheres for high-throughput single-cell transcriptome sequencing schematic diagram;

[0031] Figure 2 For bar code-based regenerable microspheres for standard chain analysis schematic diagram;

[0032] Figure 3 For library structure schematic diagram with single-cell transcriptome information;

[0033] Figure 4 For agarose gel electrophoresis diagram of standard chain verification regenerable condition;

[0034] Figure 5 For gray value diagram based on cell verification regenerable condition;

[0035] Figure 6 For fluorescence characterization diagram of nucleic acid probe modified microspheres condition. DETAILED DESCRIPTION

[0036] In order to make the personnel in the technical field better understand the application scheme, the technical scheme in the embodiment of the application will be described clearly and completely in combination with the drawings in the embodiment of the application. Obviously, the described embodiment is only a part of the embodiment of the application, not all. Based on the embodiment in the application, all other embodiments obtained by the person skilled in the art without creative labor should belong to the protection scope of the application.

[0037] It should be noted that the terms "first", "second" and the like in the specification and claims of the application and the above-mentioned drawings are used to distinguish similar objects, and do not necessarily indicate a specific order or sequence. It should be understood that the data thus used can be interchanged under appropriate circumstances, so that the embodiments of the application described herein can be implemented in an order other than that illustrated or described herein. In addition, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device including a series of steps or units does not necessarily limit to those steps or units clearly listed, but can include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0038] The application will be described in further detail below in combination with the drawings:

[0039] The application provides a bar code regenerable microsphere, which comprises a microsphere, a bar code probe, a capture probe, a universal primer probe and a strand displacement probe in terms of structure.

[0040] The bar code probe is coupled to the microsphere.

[0041] The bar code probe is provided with a universal primer hybridization region, a cell bar code region and a capture probe hybridization region; the universal primer hybridization region is used for complementary pairing with the universal primer probe, the cell bar code region is used for cell determination, and the capture probe hybridization region is used for complementary pairing with the capture probe.

[0042] The capture probe is provided with a bar code probe hybridization region, a strand displacement probe hybridization region and a capture region; the bar code probe hybridization region is used for complementary pairing with the capture probe hybridization region on the bar code probe, the strand displacement probe hybridization region is used for complementary pairing with the strand displacement probe, and the capture region is used for capturing mRNA in cells.

[0043] The universal primer probe and the capture probe are used for generating a full-length capture probe containing a universal primer probe sequence, a cell bar code sequence and a capture probe sequence through hybridization to the bar code probe and then polymerization and ligation reaction.

[0044] The strand displacement probe is used for complementary pairing with the strand displacement probe hybridization region on the capture probe and performing a polymerization strand displacement reaction to displace and release the full-length capture probe.

[0045] The universal primer described above is designed for a specific fragment of a certain gene or a specific gene and can amplify a specific DNA fragment. In the bar code regenerable microsphere, these DNA fragments can be used as coding information for product anti-counterfeiting and traceability. The universal primer has the characteristics of strong universality and high amplification efficiency, can obtain a large amount of amplification product in a short time, and is helpful to improve the detection efficiency and accuracy.

[0046] The cell bar code described above is also called a cell barcode or a single-cell barcode, which is a method of labeling and tracking at the single-cell level. By introducing unique barcode sequences on the cell surface or inside, a large number of cells can be analyzed in parallel without the need for individual operation on each cell. The microsphere containing the barcode sequence is mixed with the cells, and the microsphere is attached to the cell surface or inside through a specific method (such as physical adsorption, chemical crosslinking or biological coupling). In this way, each cell obtains a unique barcode label.

[0047] The "hybridization region" described above refers to the region involved in nucleic acid (such as DNA or RNA) hybridization during the process of biological labeling, detection or analysis using microspheres. It usually refers to the region where two complementary nucleic acid chains (such as DNA or RNA) bind to form a double strand. This binding is achieved through base pairing (A-T, C-G). Barcode regenerable microspheres are used for cell labeling, and the barcode (which may be a DNA or RNA sequence) on the microspheres may hybridize with specific nucleic acid sequences in the cell, thereby achieving cell labeling and tracking.

[0048] The "strand displacement region" described above refers to a specific region designed inside or on the surface of the microsphere, which can achieve information encoding, decoding or updating through strand displacement reaction. This design may make the microsphere not only have the function of encoding anti-counterfeiting, but also be able to realize dynamic updating or re-reading of information through strand displacement reaction, thereby improving the application flexibility and reliability of the microsphere. Strand displacement reaction (Strand Displacement Reaction, SDR) is a commonly used molecular biology technique for studying DNA recombination and repair mechanisms.

[0049] The "molecular barcode" described above refers to the main means of information storage and encoding, which is designed to have specific molecular barcodes inside or on the surface of the microsphere. These barcodes can be read or updated through chemical reactions, biological recognition or physical stimulation, and by designing different molecular sequences or structures, unique molecular barcodes can be formed inside or on the surface of the microsphere. Using specific identification techniques (such as fluorescence microscopy, mass spectrometry, etc.), the molecular barcode information inside the microsphere can be read.

[0050] The "capture region" described above refers to the region on the microsphere for binding or capturing specific biological molecules. In single-cell sequencing technology, the capture region usually has specific capture sequences such as cell barcodes (Barcode), molecular tags (UMI) and PolyT sequences. These sequences can recognize and bind to mRNA molecules in individual cells, thereby achieving single-cell transcriptome sequencing. The design of the capture region enables the microsphere to efficiently capture and label target biological molecules, improving the accuracy and throughput of single-cell sequencing.

[0051] The present application also discloses a preparation and regeneration method of the above-mentioned barcode regenerable microsphere, comprising the following steps:

[0052] 1) coupling the barcode probe to the microsphere by chemical reaction to obtain a barcode microsphere;

[0053] 2) hybridize the universal primer probe and the capture probe to the barcode probe, generate full-length capture probe containing universal primer probe sequence, cell barcode sequence and capture probe sequence by polymerization, ligation reaction;

[0054] 3) capture the molecules to be detected by the barcode microspheres obtained in step 2), and hybridize the strand displacement probe to the strand displacement probe hybridization region on the capture probe and perform polymerase chain reaction, displace and release the full-length capture probe capturing the molecules to be detected for library construction;

[0055] 4) regenerate the barcode microspheres by again performing the polymerization and ligation of step 2) on the barcode microspheres after step 3), and again generate full-length capture probes to realize regeneration of the barcode regenerable microspheres.

[0056] Preferably, in step 1), the cell barcode with a hybridization region at the end is obtained by split & pool method, and of course other methods can also obtain the cell barcode, such as chemical synthesis.

[0057] Specifically, as shown in the following table, the barcode regenerable microspheres of the present application are designed to include: Figure 1

[0058] 1. Designing probe sequences of the barcode regenerable microspheres for recognizing mRNA in cells

[0059] Designing nucleic acid probes (i.e. probe 1) for microsphere modification, the 3' end of probe 1 is combined with an amino group, and the amino group is used to combine with a carboxyl microsphere, and the microsphere modification includes but is not limited to carboxyl and aldehyde groups;

[0060] Designing read1 probe sequence (i.e. probe 2) for PCR amplification (PCR handle, PH), probe 2 is composed of two parts, 19 bases for read1 sequence for PCR amplification and 15 bases for hybridizing probe 1;

[0061] Designing probe sequence (i.e. probe 3) for capturing mRNA, the 5' end of probe 3 is modified with phosphate, and the 3' end is modified with thio, probe 3 is composed of four parts, 15 bases at the 5' end for hybridizing probe 1, 15 bases for hybridizing polymerase chain reaction displacement probe, 10 bases for UMI for molecular counting, and 30 bases at the 3' end for hybridizing mRNA sequence;

[0062] Designing probe sequence (i.e. probe 4) for polymerase chain reaction displacement, probe 4 is composed of 15 bases for hybridizing probe 3;

[0063] ​The polyA probe sequence (i.e. probe 5) designed for standard chain verification consists of two parts: a 20-base polyA at the 3' end and a 15-base Read2 sequence at the 5' end for PCR amplification.

[0064] The probe sequence (i.e. probe 6) designed for template conversion in cell samples consists of two parts: a Read2 sequence at the 5' end for PCR amplification and a LNA modification at the 3' end.

[0065] 2. Regeneration of barcode regenerable microspheres

[0066] As shown in Figure 1 , incubate the functionalized microspheres with probe 1 at a final concentration of 10 μM. Incubate probe 2 and probe 3 with the microspheres modified with probe 1, ready for use.

[0067] Use DNA polymerase and DNA ligase to fill in the gap and form a complete capture probe for subsequent standard chain and cell sample verification.

[0068] As shown in Figure 2 , use the functionalized and modified barcode microspheres for standard chain verification. In the presence of probe 5, probe 3 will bind and hybridize stably, and in the presence of DNA polymerase, the complete capture probe will dissociate from the barcode microspheres, allowing the cell barcode on the microspheres to be regenerated for use.

[0069] Subsequent PCR amplification of the dissociated probe is performed, and the size of the amplified product band is verified by agarose gel electrophoresis. After completing the first round of verification, repeat the above operations to verify the regenerability of the barcode microspheres.

[0070] 3. Application of barcode regenerable microspheres in single-cell omics analysis

[0071] As shown in Figure 1 , use the functionalized and modified barcode regenerable microspheres for cell sample verification. During the lysis process, the probe 5 sequence on the microspheres will capture mRNA molecules, and during the reverse transcription process, the microspheres will capture the genetic information in the cells. In the presence of probe 4, probe 3 will bind and hybridize stably, and in the presence of DNA polymerase, the complete capture probe will dissociate from the barcode microspheres, allowing the cell barcode on the microspheres to be regenerated for use. The dissociated probe is subjected to PCR amplification, as shown in Figure 3 , and library construction and sequencing are used to analyze the genetic information in the cells.

[0072] The application will be further described in connection with the following detailed description of specific embodiments. It should be understood that these embodiments are intended to illustrate the application and are not intended to limit its scope. Furthermore, it should be understood that various modifications and changes can be made to the application by those skilled in the art upon reading the disclosure set forth in the present application and such modifications and changes are intended to fall within the scope of the appended claims.

[0073] The following examples use apparatus and equipment that are conventional in the art. The experimental methods in the following examples, unless otherwise specified, are generally conducted under conventional conditions, or under conditions recommended by the manufacturer. The following examples use various raw materials, unless otherwise specified, all use conventional commercially available products, the specifications are conventional in the art. In the specification of the present application and the following examples, unless otherwise specified, "%" means mass percent, and the ratio means mass ratio.

[0074] 1. Design of nucleic acid probe molecules and their analogs:

[0075] In order to realize the barcoding regenerable microspheres for high-throughput single cell analysis, the corresponding nucleic acid probes are designed.

[0076] In the process of designing probe 1, according to the characteristics of carboxyl and amino groups that can be quickly coupled, one end of the amino group is combined with the carboxyl microspheres; 19 bases are designed as barcode regions, in order to determine the subsequent spatial position; In order to reduce the steric hindrance, the spacer region is designed between the microspheres and the functional region; At the same time, in order to characterize the microspheres combined with the probe, the other end is designed with a fluorescent group, and the combination can be known by the fluorescence intensity. The sequence of the designed probe 1 is shown as SCSEQ NO. 1 in Table 1.

[0077] In the process of designing probe 2, according to the primer required in the process of PCR amplification, read1 region is designed; For hybridizing probe 1, hybridization region is designed. The sequence of the designed probe 2 is shown as SCSEQ NO. 2 in Table 1.

[0078] In the process of designing probe 3, the 5' end is designed as phosphate modification, which can be connected with the probe, and the other end is designed as the capture region for hybridizing with mRNA, in order to prevent enzyme cutting, the 3' end is designed as thio modification. The sequence of the designed probe 3 is shown as SCSEQ NO. 3 in Table 1.

[0079] In the process of designing probe 4, 15 bases are designed as stable hybridization. The sequence of the designed probe 4 is shown as SCSEQ NO. 4 in Table 1.

[0080] In the process of designing probe 5, according to the primer required in the process of PCR amplification, read2 region is designed, and polyA sequence is designed for hybridizing probe 3. The sequence of the designed probe 5 is shown as SCSEQ NO. 5 in Table 1.

[0081] In the process of designing the probe 6, according to the primer required in the PCR amplification process, the read2 region is designed for hybridizing the reverse transcription product, and the LNA sequence is designed at the 3' end. The sequence of the designed probe 6 is shown as SC SEQ NO. 6 in Table 1.

[0082] Table 1 Sequence table

[0083]

[0084] The above designed probes are simulated by using NUPACK software. The simulation results show that no secondary structure is formed in the simulation of the six probes, and the pairing and hybridization efficiency of probe 1, probe 2 and probe 3 is higher. The pairing and hybridization efficiency of probe 3 and probe 4 and 5 is higher.

[0085] The above probe 1, probe 2, probe 3, probe 4, probe 5 and probe 6 are synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd. and used in the following experiments.

[0086] 2, Preparation of barcoded microspheres modified with probe 1, probe 2 and probe 3:

[0087] The probe 1 with a concentration of 10 μM is combined with the carboxyl microspheres, and the overnight shaking incubation is carried out at room temperature. The surface of the microspheres is cleaned with 1×PBST solution (containing 1% BSA) to remove the non-specifically adsorbed DNA. 20 μL of probe 2 and probe 3 with a concentration of 10 μM are hybridized with probe 1 at 37°C for 30 min. The surface of the microspheres is cleaned with 1×PBST solution (containing 1% BSA) to remove the unreacted and non-specifically adsorbed DNA. Next, T4 DNA ligase and T4 DNA polymerase are incubated with the microspheres at 37°C for 2 h for gap-filling, and the probe 1 and probe 3 are connected for the reproducible use of the barcoded microspheres.

[0088] 3, Barcoded reproducible microspheres for standard chain verification

[0089] The probe 5 with a concentration of 10 μM is reacted with the barcoded microspheres prepared in step 2, and after 37°C reaction for 2 h, the surface of the microspheres is cleaned with 1×PBST solution (containing 1% BSA) to remove the unreacted and non-specifically adsorbed DNA. Finally, the standard chain with a barcode is obtained by DNA polymerase chain displacement, so that the microspheres can be reused. As shown in Figure 4 , three rounds of verification are carried out, and the reproducibility is characterized by PCR and agarose gel electrophoresis.

[0090] 4, Barcoded reproducible microspheres for tumor cell sample verification

[0091] Select tumor cell MCF-7, prepare cell suspension of suitable concentration, and carry out cell activity analysis, and the effect is best when the rate of living cells is greater than 90%. After mixing the cell suspension and the bar code microspheres uniformly, add cell lysis solution, and carry out 30 min of shaking reaction at 37 DEG C, carry out cell lysis and mRNA capture. Wash the microspheres with 6xSSC solution, and add reverse transcription reagent to carry out reverse transcription and template conversion, 6 h of reaction at 42 DEG C, mRNA reverse transcription becomes cDNA. Add 10 μM concentration probe 4 to make it hybridize with probe 3. Finally, obtain cDNA sequence through DNA polymerase chain displacement, and repeat it, as shown in the formula (I), carry out three rounds of renewable ability verification, carry out PCR and agarose gel electrophoresis to characterize its renewable ability, carry out second-generation sequencing to determine its transcriptome information, and finally obtain library display, and the cDNA library fragment is concentrated in 500-2000 bp, and the sequencing DNA library fragment is concentrated near 400 bp, and there is no interference of miscellaneous band. Figure 5

[0092] The above is only for illustrating the technical idea of the present application, and cannot limit the protection scope of the present application, and any modification made according to the technical idea of the present application on the basis of the technical scheme falls within the protection scope of the claims of the present application.​

Claims

1. A barcoded reproducible microsphere, characterized in that, The microspheres, the barcode probe, the capture probe, the universal primer probe and the strand displacement probe are included. The barcode probe is coupled with the microspheres. The barcode probe is provided with a universal primer hybridization region, a cell barcode region and a capture probe hybridization region; the universal primer hybridization region is used for complementary pairing with the universal primer probe, the cell barcode region is used for cell determination, and the capture probe hybridization region is used for complementary pairing with the capture probe. The capture probe is provided with a barcode probe hybridization region, a strand displacement probe hybridization region and a capture region; the barcode probe hybridization region is used for complementary pairing with the capture probe hybridization region on the barcode probe, the strand displacement probe hybridization region is used for complementary pairing with the strand displacement probe, and the capture region is used for capturing mRNA in cells. The universal primer probe and the capture probe are used for generating a full-length capture probe containing a universal primer probe sequence, a cell barcode sequence and a capture probe sequence through hybridization to the barcode probe and then polymerization and ligation reactions. The strand displacement probe is used for complementary pairing with the strand displacement probe hybridization region on the capture probe and performing a polymerization strand displacement reaction to displace and release the full-length capture probe.

2. The bar-codable microsphere of claim 1, wherein, The capture probe is further provided with a molecular barcode region, and the molecular barcode is different on a single microsphere and is used for coding different molecules to be detected.

3. The bar-codable microsphere of claim 1, wherein, The material of the microspheres is selected from one or more of polystyrene, polymethacrylate, silicon dioxide and polyacrylamide; and the diameter of the microspheres is 0.1-1000 μm.

4. The bar-codable microsphere of claim 1, wherein, The surface of the microspheres is modified by a modification group, and the modification group includes one or more of a carboxyl group, an epoxy group, an alkyne group, an N-hydroxysuccinimide group, an aldehyde group and streptavidin; and the nucleic acid molecule on the barcode probe is connected to the modification group on the surface of the microspheres through a specific group, and the specific group includes one or more of an amino group, a sulfhydryl group and an epoxy group.

5. The method for preparing and regenerating the bar code reproducible microspheres according to any one of claims 1 to 4, characterized in that, The method includes the following steps: 1) coupling the barcode probe to the microspheres through a chemical reaction to obtain barcode microspheres; 2) hybridizing the universal primer probe and the capture probe to the barcode probe to generate a full-length capture probe containing a universal primer probe sequence, a cell barcode sequence and a capture probe sequence through polymerization and ligation reactions; 3) capturing a molecule to be detected by using the barcode microspheres obtained in step 2), hybridizing the strand displacement probe to the strand displacement probe hybridization region on the capture probe and performing a polymerization strand displacement reaction to displace and release a full-length capture probe capturing the molecule to be detected for library construction; 4) performing the polymerization and ligation treatment of step 2) again on the barcode microspheres treated in step 3) to generate a full-length capture probe again, so as to regenerate the barcode regenerable microspheres.

6. Use of the barcoded regenerable microspheres according to any one of claims 1 to 4 in single-cell omics analysis for non-disease diagnostic purposes, characterized in that, The method includes: mixing a cell suspension with barcode regenerable microspheres, performing cell lysis and mRNA capture, and then performing reverse transcription to convert mRNA into cDNA; then performing a polymerization strand displacement reaction to displace and release a full-length capture probe capturing the cDNA sequence, amplifying and constructing a sequencing library, and at this time, the barcode regenerable microspheres can be regenerated and used; finally, sequencing and single-cell omics analysis are performed.

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

1.

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

Images