Methods of modifying solid supports and cell capture

Abstract

The application provides a method for modifying a solid support suitable for adhering cells, comprising: contacting the solid support with a polylysine-protein factor mixture, the polylysine-protein factor mixture comprising polylysine and a protein factor, the protein factor comprising at least one of an extracellular matrix protein and a cell integrin. The method modifies the solid support, allows the solid support to capture a large number of cells, makes the profile of the cells captured by the solid support clear, and improves the cell capture efficiency of the solid support.

Description

Methods for modifying solid supports and cell capture Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to a method for modifying a solid support and capturing cells. More specifically, the present invention relates to a method for modifying a solid support, a solid support, a cell capture method, an RNA capture method, a library construction method, and a sequencing method. Background Art

[0002] Solid supports consist of solid matrix materials that are used to immobilize or support other biomolecules (such as proteins, nucleic acids, antibodies, etc.) for specific biochemical or biological experiments. These solid supports provide a stable platform that enables biomolecules to interact with other molecules in the sample. Common solid supports include: polymer matrices, porous materials (such as silicon wafers and glass slides, used to prepare microarrays such as gene chips and protein chips), metal ion affinity matrices, magnetic particles, cellulose matrices, gel matrices, and gel filtration plates.

[0003] Solid supports can be modified in the following ways: introducing specific functional groups or chemical functional groups (carboxyl, amino, sulfide, etc.) on the surface of the solid support to achieve selective recognition and fixation of cells; using specific antibodies to bind to chemical functional groups on the surface of the solid support to achieve highly specific capture and fixation of cells; using the affinity of biological molecules to specifically bind to the surface of the solid support; changing the properties and characteristics of the solid support surface through physical treatment or specific coating technology.

[0004] In biological research, solid supports are often designed for specific applications, such as capturing, immobilizing, and isolating specific cell types in single-cell sequencing for subsequent analysis and research. Current single-cell sequencing technologies place higher demands on the throughput and efficiency of cell capture using solid supports. Therefore, there is an urgent need to develop methods to modify solid supports to improve their cell capture efficiency.

[0005] Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0007] The inventors discovered that using a poly-L-lysine-modified chip (1cm*1cm) with a cell input of 10,000 or less, the cell capture efficiency reached over 80%. However, when the cell throughput was increased to 20,000, the poly-L-lysine-modified chip experienced severe diffusion, blurred cell outlines, and was unable to capture a large number of cells, seriously affecting the cell capture efficiency (see Figure 1 for specific results). To overcome this problem, the inventors added protein factors to the poly-lysine-based chip based on existing methods. This modified chip (1cm*1cm) can be loaded with more than 20,000 cells at a time, and achieved a cell capture efficiency of 90% in single-cell transcriptome analysis, effectively improving cell throughput and capture efficiency.

[0008] Based on this, the first aspect of the present invention proposes the use of polylysine and protein factors to improve the efficiency of cell capture on a solid support. The protein factors include at least one of an extracellular matrix protein and a cellular integrin. According to embodiments of the present invention, a solid support modified with polylysine and an extracellular matrix protein or a cellular integrin can capture a large number of cells, providing a clear outline of the cells captured by the solid support, thereby improving the cell capture efficiency of the solid support. The solid support comprises a plurality of microspots, each of which is coupled to an oligonucleotide probe, each of which carries a specific primer with a spatial barcode. In embodiments of the present invention, these microspots can be used as a probe array at specific locations to capture mRNA at corresponding locations in cells in situ. When cells interact with these microspots, specific mRNAs bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, thereby enabling clear observation of the cell outline.

[0009] In a second aspect, the present invention provides a method for modifying a solid support. According to an embodiment of the present invention, the method comprises contacting the solid support with a polylysine-protein factor mixture, wherein the polylysine-protein factor mixture comprises polylysine and a protein factor, wherein the protein factor comprises at least one of an extracellular matrix protein and a cellular integrin. Modifying the solid support according to the method of the embodiment of the present invention allows the solid support to capture a large number of cells, providing clear outlines of the cells captured by the solid support, and improving the cell capture efficiency of the solid support. The solid support comprises a plurality of microspots, each of which is coupled to an oligonucleotide probe, each of which carries a specific primer with a spatial barcode. In an embodiment of the present invention, these microspots can be used as a probe array at specific locations to capture mRNA in situ at corresponding locations in cells. When cells interact with these microspots, specific mRNAs bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcodes on the detected mRNA can be restored to the cells, thereby enabling clear observation of the cell outlines.

[0010] The third aspect of the present invention provides a solid support. According to an embodiment of the present invention, the solid support is obtained after modification using the method of the second aspect of the present invention. The solid support according to the embodiment of the present invention can capture a large number of cells, the captured cells have clear outlines, and the cell capture efficiency is improved. Among them, the solid support contains a plurality of micro-dots, each micro-dot is coupled with an oligonucleotide probe, and these probes have specific primers with spatial barcodes. In an embodiment of the present invention, these micro-dots can be used as probe arrays at specific positions to capture mRNA at corresponding positions of cells in situ. When cells interact with these micro-dots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, so that clear observation of the cell outline can be achieved.

[0011] In a fourth aspect, the present invention provides a solid support. According to an embodiment of the present invention, the solid support comprises: a basal layer and polylysine and a protein factor, wherein the polylysine and protein factor are arranged on the upper surface of the basal layer, and the protein factor comprises at least one of an extracellular matrix protein and a cellular integrin. The solid support according to an embodiment of the present invention can capture a large number of cells, the captured cells have clear outlines, and the cell capture efficiency is improved. The solid support comprises a plurality of microdots, each of which is coupled to an oligonucleotide probe, and these probes carry specific primers with spatial barcodes. In an embodiment of the present invention, these microdots can be used as probe arrays at specific locations for in situ capture of mRNA at corresponding locations of cells. When cells interact with these microdots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, enabling clear observation of the cell outline.

[0012] In its fifth aspect, the present invention provides a method for cell capture. According to an embodiment of the present invention, the method comprises: modifying a solid support using the method described in the second aspect of the present invention; and performing cell capture on a sample to be captured using the modified solid support. Cell capture using the method of an embodiment of the present invention can significantly improve cell capture efficiency and capture cells with clear outlines.

[0013] In its sixth aspect, the present invention provides a method for RNA capture. According to an embodiment of the present invention, the method comprises: capturing cells from a sample using the method described in the fifth aspect of the present invention; and permeabilizing the captured cells to obtain RNA from the sample. Capturing RNA using the method of an embodiment of the present invention can significantly improve RNA capture efficiency by increasing the throughput of captured cells.

[0014] In a seventh aspect, the present invention provides a library construction method comprising: obtaining RNA from a test sample using the method of the sixth aspect; and performing reverse transcription and amplification on the RNA from the test sample to obtain a sequencing library. Library construction according to the method of an embodiment of the present invention can significantly improve the throughput of the sequencing library by increasing the throughput of captured cells and thereby increasing the number of sequenced fragments.

[0015] In an eighth aspect, the present invention provides a sequencing method comprising: constructing a sequencing library using the method of the seventh aspect; and performing sequencing on the sequencing library to obtain sequencing results. Sequencing according to the methods of embodiments of the present invention can significantly improve the throughput of sequencing results by increasing the number of sequenced fragments by increasing the throughput of captured cells.

[0016] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG1 is a diagram showing cell capture on a chip modified with poly-lysine when the cell amount is 1W and 2W (1W in FIG1 refers to 10,000 cells, 2W refers to 20,000 cells, and PLL refers to poly-lysine).

[0018] FIG2 is a diagram of cell capture in Example 1 in which polylysine is combined with different extracellular matrix proteins (fibronectin, vitronectin, laminin) and integrins. In FIG2 , PLL and Pll both refer to polylysine.

[0019] Figure 3 is a comparison chart of the cell capture rates of the chip modified with polylysine only, the chip modified with fibronectin only, the chip modified with laminin only, the chip modified with fibronectin and laminin only in Example 2, the chip modified with polylysine combined with fibronectin of the present invention, the chip modified with polylysine combined with laminin of the present invention, and the chip modified with polylysine combined with fibronectin and laminin of the present invention. PLL in Figure 3 refers to polylysine.

[0020] FIG4 is a graph comparing the cell capture rates of the chip modified with polylysine combined with concanavalin in a comparative example and the chip modified with polylysine combined with fibronectin in the present invention. DETAILED DESCRIPTION

[0021] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0022] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0023] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in the present invention, all other technical and scientific terms used in the present invention have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0024] In the present invention, the term "comprise" or "include" is an open expression, that is, it includes the content specified in the present invention, but does not exclude other aspects of the content.

[0025] In the present invention, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0026] The present invention proposes the use of polylysine and extracellular matrix proteins or cellular integrins in improving the efficiency of cell capture on a solid support, a method for modifying a solid support, a solid support, a cell capture method, an RNA capture method, a library construction method, and a sequencing method, each of which will be described in detail below.

[0027] use

[0028] In its first aspect, the present invention proposes the use of polylysine and a protein factor to improve the efficiency of cell capture on a solid support. The protein factor comprises at least one of an extracellular matrix protein and a cellular integrin. According to embodiments of the present invention, a solid support modified with polylysine and either an extracellular matrix protein or a cellular integrin can capture a large number of cells, resulting in clear cell outlines on the solid support and improving the cell capture efficiency of the solid support. Furthermore, the solid support is suitable for adherent cells. The solid support comprises a plurality of microspots, each coupled to an oligonucleotide probe with a specific primer containing a spatial barcode. In embodiments of the present invention, these microspots can be used as a probe array at specific locations to capture mRNA in situ at corresponding locations in cells. When cells interact with these microspots, specific mRNA binds to the corresponding probe. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, thereby enabling clear observation of the cell outline.

[0029] According to an embodiment of the present invention, the extracellular matrix protein includes at least one of laminin, vitronectin, fibronectin, collagen, elastin, macromolecular glycoproteins, adhesion proteins, and cellulose. According to an embodiment of the present invention, a solid support modified with polylysine and extracellular matrix proteins or cellular integrins can capture a large number of cells, making the cell outlines captured by the solid support clearer and further improving the cell capture efficiency of the solid support. The solid support contains multiple microdots, each of which is coupled with an oligonucleotide probe, and these probes have specific primers with spatial barcodes. In an embodiment of the present invention, these microdots can be used as probe arrays at specific locations to capture mRNA at corresponding locations in cells in situ. When cells interact with these microdots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, thereby achieving clear observation of the cell outline.

[0030] According to an embodiment of the present invention, the extracellular matrix protein includes at least one of laminin, vitronectin and fibronectin. The solid support modified with polylysine and laminin, vitronectin or fibronectin according to an embodiment of the present invention can capture a large number of cells, making the cell outlines captured by the solid support clearer, and further improving the cell capture efficiency of the solid support. Wherein, the solid support contains a plurality of micro-dots, each of which is coupled with an oligonucleotide probe, and these probes are provided with specific primers of spatial barcodes. In an embodiment of the present invention, these micro-dots can be used as probe arrays at specific positions for in situ capture of mRNA at corresponding positions of cells. When cells interact with these micro-dots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, thereby achieving clear observation of the cell outline.

[0031] According to an embodiment of the present invention, the cell integrin includes at least one of integrin and adhesion protein. The solid support modified with polylysine and cell integrin according to an embodiment of the present invention can capture a large number of cells, making the cell outlines captured by the solid support clearer, and further improving the cell capture efficiency of the solid support. Among them, the solid support contains a plurality of micro-dots, each micro-dot is coupled with an oligonucleotide probe, and these probes are provided with specific primers of spatial barcodes. In an embodiment of the present invention, these micro-dots can be used as probe arrays at specific positions for in situ capture of mRNA at corresponding positions of cells. When cells interact with these micro-dots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, thereby achieving clear observation of the cell outline.

[0032] According to an embodiment of the present invention, the cell integrin includes integrin. The solid support modified with polylysine and integrin according to an embodiment of the present invention can capture a large number of cells, making the outline of the cells captured by the solid support clearer, and further improving the cell capture efficiency of the solid support. Among them, the solid support contains a plurality of microdots, each microdot is coupled with an oligonucleotide probe, and these probes have specific primers with spatial barcodes. In an embodiment of the present invention, these microdots can be used as probe arrays at specific positions for in situ capture of mRNA at corresponding positions of cells. When cells interact with these microdots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, thereby achieving clear observation of the cell outline.

[0033] According to an embodiment of the present invention, the solid support comprises at least one of a gel matrix, magnetic particles, a cellulose matrix, a metal ion affinity matrix, a polymer matrix, a porous material and a gel filtration plate.

[0034] According to an embodiment of the present invention, the porous material includes a microarray made of a silicon wafer, a glass wafer or a polymer material.

[0035] According to an embodiment of the present invention, the surface of the solid support includes nucleic acid molecules.

[0036] According to an embodiment of the present invention, the nucleic acid molecule is labeled, and the label includes but is not limited to at least one of a radioactive label, a fluorescent label, an enzyme label, a biotin label and an immune label.

[0037] It should be noted that radiolabeling includes the use of radioisotopes (such as ^32P or ^ 35S) labeling of nucleic acids. These isotopes emit radiation and can be detected by darkroom photography or scintillation counters. Fluorescent labeling is a commonly used nucleic acid labeling method. By binding fluorescent dyes or fluorescent proteins (such as fluorescein or green fluorescent protein) to nucleic acids, labeled nucleic acids can be detected and observed using techniques such as fluorescence microscopy or flow cytometry. Enzyme labeling is the binding of specific enzymes (such as horseradish peroxidase (HRP) or alkaline phosphatase (AP)) to nucleic acids. These enzymes can react with dyes to produce visible color or fluorescent signals, thereby enabling the detection of nucleic acids. Biotin labeling is a small molecule that can bind to nucleic acids. Biotin-labeled nucleic acids can interact with substances with affinity (such as fluorescent dyes or enzymes), thereby enabling the detection of nucleic acids. Immunolabeling is the labeling of nucleic acids using the specific binding of antibodies to nucleic acids. By binding antibodies to markers such as fluorescent dyes, enzymes or radioactive isotopes, nucleic acids can be detected and visualized. This labeling method is often used in techniques such as immunohistochemistry and immunoblotting.

[0038] According to an embodiment of the present invention, the nucleic acid molecule is labeled, and the label includes but is not limited to at least one of a fluorescent dye and an enzyme label.

[0039] According to an embodiment of the present invention, the nucleic acid molecule is modified, and the modification includes but is not limited to at least one of antisense modification, modification catalyzed by a modification enzyme, chemical cross-linking, magnetic modification, nanopore array modification and optical modification.

[0040] It should be explained in the present invention that antisense modification refers to modification by changing the base sequence or nucleotide pairing mode in the nucleic acid molecule. For example, antisense oligonucleotides or antisense RNA can be used to interact with specific sequences in the target nucleic acid molecule to regulate its structure and function; modification catalyzed by modifying enzymes refers to the use of specific modifying enzymes to catalyze reactions to introduce modifying groups or chemicals into specific positions in the nucleic acid molecule. For example, DNA methyltransferase can introduce methyl groups on DNA molecules, thereby changing the methylation pattern of DNA; chemical crosslinking is the process of connecting two or more parts of a nucleic acid molecule together through a chemical reaction. This modification method can be used to construct molecular complexes, stabilize Determine the structure of nucleic acids or prepare nucleic acid nanostructures; magnetic modification is the introduction of magnetic materials into nucleic acid molecules to give them magnetic properties. This modification method is often used in applications such as magnetic separation and magnetic resonance imaging, and can achieve rapid separation and detection of nucleic acids; nanopore array modification refers to the introduction of specific chemical modifiers on the surface of the nanopore structure to enable it to interact with nucleic acid molecules. This modification method is often used in nanopore sequencing technology to achieve high-throughput sequencing of single nucleic acid molecules; optical modification is the introduction of fluorescent dyes, quantum dots or other optical markers into nucleic acid molecules to achieve fluorescent detection, imaging or tracking of nucleic acids. This modification method is often used in biological fluorescence microscopy technology and flow cytometry.

[0041] According to an embodiment of the present invention, the solid support is a sequencing chip. The sequencing chip modified with polylysine and integrin according to an embodiment of the present invention can capture a large number of cells, so that the cell outlines captured by the sequencing chip are clear, and the cell capture efficiency of the sequencing chip is improved. Among them, the sequencing chip contains multiple microdots, each of which is coupled with an oligonucleotide probe, and these probes are specific primers with spatial barcodes. In an embodiment of the present invention, these microdots can be used as probe arrays at specific positions for in situ capture of mRNA at corresponding positions of cells. When cells interact with these microdots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, thereby achieving clear observation of the cell outline.

[0042] Methods for modifying solid supports

[0043] In a second aspect, the present invention provides a method for modifying a solid support. According to an embodiment of the present invention, the method comprises contacting the solid support with a polylysine-protein factor mixture, wherein the polylysine-protein factor mixture comprises polylysine and a protein factor, wherein the protein factor comprises at least one of an extracellular matrix protein and a cellular integrin. Modifying the solid support according to the method of the embodiment of the present invention allows the solid support to capture a large number of cells, providing clear outlines of the cells captured by the solid support, and improving the cell capture efficiency of the solid support. The solid support comprises a plurality of microspots, each of which is coupled to an oligonucleotide probe, each of which carries a specific primer with a spatial barcode. In an embodiment of the present invention, these microspots can be used as a probe array at specific locations to capture mRNA in situ at corresponding locations in cells. When cells interact with these microspots, specific mRNAs bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcodes on the detected mRNA can be restored to the cells, thereby enabling clear observation of the cell outlines.

[0044] According to an embodiment of the present invention, the contact treatment is performed by dropping the poly-lysine-protein factor mixture onto the solid support.

[0045] According to an embodiment of the present invention, the molecular weight of the polylysine is not less than 70,000 Daltons. Preferably, the molecular weight of the polylysine is 70,000 to 300,000 Daltons. More preferably, the molecular weight of the polylysine is 70,000 to 150,000 Daltons.

[0046] According to an embodiment of the present invention, in the polylysine-protein factor mixture, the mass ratio of the polylysine to the protein factor is 1:1 to 1:10000. The polylysine-protein factor mixture according to the embodiment of the present invention is used to modify the solid phase support, so that the modified solid phase support can capture a larger number of cells, make the cell outlines captured by the modified solid phase support clearer, and further improve the cell capture efficiency of the solid phase support. Among them, the solid phase support contains multiple micro-dots, each micro-dot is coupled with an oligonucleotide probe, and these probes have specific primers with spatial barcodes. In an embodiment of the present invention, these micro-dots can be used as probe arrays at specific locations for in situ capture of mRNA at corresponding locations of cells. When cells interact with these micro-dots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, thereby achieving clear observation of the cell outline.

[0047] According to an embodiment of the present invention, in the polylysine-protein factor mixture, the mass ratio of the polylysine to the protein factor is 1:25 to 1:50. The polylysine-protein factor mixture according to an embodiment of the present invention is used to modify the solid phase support, so that the modified solid phase support can capture cells in larger quantities, so that the cell outlines captured by the modified solid phase support are clearer, and the cell capture efficiency of the solid phase support is further improved. Among them, the solid phase support contains a plurality of microdots, each microdot is coupled with an oligonucleotide probe, and these probes are specific primers with spatial barcodes. In an embodiment of the present invention, these microdots can be used as probe arrays at specific positions for in situ capture of mRNA at corresponding positions of cells. When cells interact with these microdots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, thereby achieving clear observation of the cell outline.

[0048] According to an embodiment of the present invention, in the polylysine-protein factor mixture, the mass ratio of the polylysine and the protein factor is 1:1, 1:10, 1:25, 1:50, 1:100, 1:250, 1:500, 1:1000 or 1:10000. The polylysine-protein factor mixture according to the embodiment of the present invention is modified to form a solid support, so that the modified solid support can capture cells in larger quantities, and the cell outlines captured by the modified solid support are clearer, thereby further improving the cell capture efficiency of the solid support. Wherein, the solid support comprises a plurality of microdots, each of which is coupled to an oligonucleotide probe, and these probes are provided with specific primers of spatial barcodes. In an embodiment of the present invention, these microdots can be used as probe arrays at specific locations for in situ capture of mRNA at corresponding positions of cells. When cells interact with these microdots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using specific algorithms, the spatial barcodes on the detected mRNA can be restored to the cells, enabling clear observation of the cell outline.

[0049] According to an embodiment of the present invention, in the polylysine-protein factor mixture, the mass ratio of the polylysine to the protein factor is 1:25 or 1:50. The polylysine-protein factor mixture according to the embodiment of the present invention is used to modify the solid phase support, so that the modified solid phase support can capture cells in larger quantities, make the cell contours captured by the modified solid phase support clearer, and further improve the cell capture efficiency of the solid phase support. Among them, the solid phase support contains a plurality of microdots, each microdot is coupled with an oligonucleotide probe, and these probes are provided with specific primers of spatial barcodes. In an embodiment of the present invention, these microdots can be used as probe arrays at specific positions for in situ capture of mRNA at corresponding positions of cells. When cells interact with these microdots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, thereby achieving clear observation of the cell contour.

[0050] According to an embodiment of the present invention, the dropping treatment further includes incubating the dropped product, and the incubation is performed at a temperature of 23° C.-37° C. and a rotation speed of 50 rpm-250 rpm for not less than 60 minutes.

[0051] According to an embodiment of the present invention, the dropping treatment further includes incubating the dropped product, and the incubation is performed at a temperature of 23° C. and a rotation speed of 50 rpm for 60 minutes.

[0052] According to an embodiment of the present invention, the extracellular matrix protein includes at least one of laminin, vitronectin, fibronectin, collagen, elastin, macromolecular glycoprotein, adhesion protein and cellulose. The solid support modified with polylysine and extracellular matrix protein according to an embodiment of the present invention can capture a large number of cells, making the cell outlines captured by the modified solid support clearer, further improving the cell capture efficiency of the solid support. Wherein, the solid support contains multiple micro-dots, each of which is coupled with an oligonucleotide probe, and these probes are equipped with specific primers with spatial barcodes. In an embodiment of the present invention, these micro-dots can be used as probe arrays at specific locations for in situ capture of mRNA at corresponding locations of cells. When cells interact with these micro-dots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, thereby achieving clear observation of the cell outline.

[0053] According to an embodiment of the present invention, the extracellular matrix protein includes at least one of laminin, vitronectin and fibronectin. The solid support modified with polylysine and laminin, vitronectin or fibronectin according to an embodiment of the present invention can further capture cells in large quantities, making the cell outlines captured by the modified solid support clearer, and further improving the cell capture efficiency of the solid support. Wherein, the solid support comprises a plurality of microdots, each of which is coupled with an oligonucleotide probe, and these probes are provided with specific primers of spatial barcodes. In an embodiment of the present invention, these microdots can be used as probe arrays at specific positions for in situ capture of mRNA at corresponding positions of cells. When cells interact with these microdots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, thereby achieving clear observation of the cell outline.

[0054] According to an embodiment of the present invention, the cell integrin includes at least one of integrin and adhesion protein. The solid support modified with polylysine and cell integrin according to an embodiment of the present invention can capture a large number of cells, so that the cell outline captured by the modified solid support is clearer, and the cell capture efficiency of the solid support is further improved. Among them, the solid support contains a plurality of micro-dots, each micro-dot is coupled with an oligonucleotide probe, and these probes are provided with specific primers of spatial barcodes. In an embodiment of the present invention, these micro-dots can be used as probe arrays at specific positions for in situ capture of mRNA at corresponding positions of cells. When cells interact with these micro-dots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, thereby achieving clear observation of the cell outline.

[0055] According to an embodiment of the present invention, the cell integrin includes integrin. The solid support modified with polylysine and integrin according to an embodiment of the present invention can capture a large number of cells, so that the cell outline captured by the modified solid support is clearer, and the cell capture efficiency of the solid support is further improved. Among them, the solid support contains a plurality of microdots, each microdot is coupled with an oligonucleotide probe, and these probes are provided with specific primers of spatial barcodes. In an embodiment of the present invention, these microdots can be used as probe arrays at specific positions for in situ capture of mRNA at corresponding positions of cells. When cells interact with these microdots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, thereby achieving clear observation of the cell outline.

[0056] According to an embodiment of the present invention, the solid support comprises at least one of a gel matrix, magnetic particles, a cellulose matrix, a metal ion affinity matrix, a polymer matrix, a porous material and a gel filtration plate.

[0057] According to an embodiment of the present invention, the porous material includes a microarray made of a silicon wafer, a glass wafer or a polymer material.

[0058] According to an embodiment of the present invention, the surface of the solid support includes nucleic acid molecules.

[0059] According to an embodiment of the present invention, the label includes at least one of a radioactive label, a fluorescent label, an enzyme label, a biotin label and an immune label.

[0060] According to an embodiment of the present invention, the nucleic acid molecule is modified, and the modification includes at least one of antisense modification, modification catalyzed by a modification enzyme, chemical cross-linking, magnetic modification, nanopore array modification and optical modification.

[0061] Solid support

[0062] The third aspect of the present invention provides a solid support. According to an embodiment of the present invention, the solid support is obtained after modification using the method of the second aspect of the present invention. The solid support according to the embodiment of the present invention can capture a large number of cells, the captured cells have clear outlines, and the cell capture efficiency is improved. Among them, the solid support contains a plurality of micro-dots, each micro-dot is coupled with an oligonucleotide probe, and these probes have specific primers with spatial barcodes. In an embodiment of the present invention, these micro-dots can be used as probe arrays at specific positions to capture mRNA at corresponding positions of cells in situ. When cells interact with these micro-dots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, so that clear observation of the cell outline can be achieved.

[0063] Solid support

[0064] In a fourth aspect, the present invention provides a solid support. According to an embodiment of the present invention, the solid support comprises: a basal layer and polylysine and a protein factor, wherein the polylysine and protein factor are arranged on the upper surface of the basal layer, and the protein factor comprises at least one of an extracellular matrix protein and a cellular integrin. The solid support according to an embodiment of the present invention can capture a large number of cells, the captured cells have clear outlines, and the cell capture efficiency is improved. The solid support comprises a plurality of microdots, each of which is coupled to an oligonucleotide probe, and these probes carry specific primers with spatial barcodes. In an embodiment of the present invention, these microdots can be used as probe arrays at specific locations for in situ capture of mRNA at corresponding locations of cells. When cells interact with these microdots, specific mRNA will bind to the corresponding probes. The captured mRNA can be further sequenced to obtain its sequence information. By using a specific algorithm, the spatial barcode on the detected mRNA can be restored to the cell, enabling clear observation of the cell outline.

[0065] According to an embodiment of the present invention, the mass ratio of the polylysine to the protein factor is 1:1 to 1:10000.

[0066] According to an embodiment of the present invention, the mass ratio of the polylysine to the protein factor is 1:25 to 1:50.

[0067] According to an embodiment of the present invention, the mass ratio of polylysine to protein factor is 1:1, 1:10, 1:25, 1:50, 1:100, 1:250, 1:500, 1:1000 or 1:10000.

[0068] According to an embodiment of the present invention, the mass ratio of polylysine to protein factor is 1:25 or 1:50.

[0069] According to an embodiment of the present invention, the distribution density of the poly-lysine on the surface of the base layer is 1.3x10 -10 ~5.7x10 -10 mol / cm 2 .

[0070] According to an embodiment of the present invention, the distribution density of the protein factor on the surface of the basal layer is 1.35x10 -10 ~1.35x10 -6 mol / cm 2 .

[0071] Cell capture method

[0072] In its fifth aspect, the present invention provides a method for cell capture. According to an embodiment of the present invention, the method comprises: modifying a solid support using the method described in the second aspect of the present invention; and performing cell capture on a sample to be captured using the modified solid support. Cell capture using the method of an embodiment of the present invention can significantly improve cell capture efficiency and capture cells with clear outlines.

[0073] According to an embodiment of the present invention, the sample to be captured includes at least one of a single cell sample and a tissue section.

[0074] According to an embodiment of the present invention, the single cell is provided in the form of a single cell suspension.

[0075] According to an embodiment of the present invention, the sample to be captured is a single cell suspension, and the cell capture process includes: dropping the single cell suspension onto the surface of the modified solid support; and fixing the modified solid support to which the single cell suspension is dropped.

[0076] RNA capture method

[0077] In its sixth aspect, the present invention provides a method for RNA capture. According to an embodiment of the present invention, the method comprises: capturing cells from a sample using the method described in the fifth aspect of the present invention; and permeabilizing the captured cells to obtain RNA from the sample. Capturing RNA using the method of an embodiment of the present invention can significantly improve RNA capture efficiency by increasing the throughput of captured cells.

[0078] Database construction method

[0079] In a seventh aspect, the present invention provides a library construction method comprising: obtaining RNA from a test sample using the method of the sixth aspect; and performing reverse transcription and amplification on the RNA from the test sample to obtain a sequencing library. Library construction according to the method of an embodiment of the present invention can significantly improve the throughput of the sequencing library by increasing the throughput of captured cells and thereby increasing the number of sequenced fragments.

[0080] Sequencing methods

[0081] In an eighth aspect, the present invention provides a sequencing method comprising: constructing a sequencing library using the method of the seventh aspect; and performing sequencing on the sequencing library to obtain sequencing results. Sequencing according to the methods of embodiments of the present invention can significantly improve the throughput of sequencing results by increasing the number of sequenced fragments by increasing the throughput of captured cells.

[0082] According to an embodiment of the present invention, the sequencing process is performed on the MGISEQ-2000 platform.

[0083] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0084] Example 1: Detection of polylysine combined with different extracellular matrix proteins (fibronectin, vitronectin, laminin) and integrins

[0085] The sequence information involved in this embodiment is shown in Table 1:

[0086] Table 1: Sequence information

[0087] Note: "r" indicates that the nucleotide at the 3' adjacent position is a ribonucleotide; "+" indicates that the nucleotide at the 3' adjacent position has LNA (locked nucleotide) modification; "*" indicates phosphorothioate modification; "p" indicates phosphorylation modification; N = A, T, CorG; V = A, C or G.

[0088] 1. Chip Modification

[0089] (1) Poly-lysine solution and three different extracellular matrix proteins (fibronectin, vitronectin, laminin) and integrin were mixed at a ratio of 1:50, and added dropwise to the surface of the chip in a 24-well plate. After rapid manual mixing, the mixture was placed on a shaker at 50 rpm and incubated at 23°C for 60 min.

[0090] (2) Use a pipette to remove the solution from the well plate and wash twice with 1 mL of 1× PBS.

[0091] 2. Single Cell Fixation

[0092] (1) Take approximately 20,000 PBMC cells and prepare a cell suspension (PBS solution) according to conventional methods.

[0093] (2) Drop the prepared cell suspension onto the modified chip (the probes of the chip are connected to specific DNA fragments, including Barcode, UMI, and PolyT). After incubation at room temperature for 10 minutes, place the chip in -20℃ pre-cooled methanol for 30 minutes.

[0094] (3) The cells were permeabilized by treating with hydrochloric acid and 10% pepsin for 30 seconds.

[0095] 3. cDNA Synthesis

[0096] (1) cDNA synthesis

[0097] Prepare 200 μL of the reverse transcriptase reaction mixture shown in Table 2, apply the reaction mixture to the chip, cover thoroughly, and incubate at 42°C for 90-180 minutes. The reverse transcriptase will synthesize cDNA using the mRNA as a template and a polyT-containing primer, adding a CCC overhang to the 3' end of the cDNA strand. After hybridization and annealing of the TSO sequence (specific sequence shown in SEQ ID NO: 1) with the cDNA strand (through complementary pairing of the GGG residue at the end of the TSO sequence with the CCC overhang of the cDNA strand), the reverse transcriptase will continue to extend the cDNA strand using the TSO sequence as a template, adding a known primer adapter to the 3' end of the cDNA strand.

[0098] Table 2: Reverse transcription reaction system

[0099] (2) cDNA release

[0100] Configure the cDNA release system as shown in Table 3

[0101] Table 3: cDNA release system

[0102] Add 400 μL / well of cDNA Release Mix to the reaction wells of the above chip, cover the reaction wells with the chip with sealing film to seal, cover the plate with the cover and seal the outer ring to prevent volatilization, and react in a 55°C incubator for 3-17 hours.

[0103] (3) cDNA amplification

[0104] Prepare 200 μL of the reaction system shown in Table 4 for 3' end transcriptome sequencing library construction, divided into two PCR tubes:

[0105] Table 4: cDNA amplification system

[0106] The reaction system was placed in a PCR instrument and set to the following: 95°C for 3 min, 15 cycles (98°C for 20 s, 58°C for 20 s, 72°C for 3 min), 72°C for 5 min, and 4°C for ∞. After the reaction, magnetic beads (purchased from Novozymes) were used for purification and recovery. The dsDNA concentration was quantified using a Qubit instrument, and the length distribution of the cDNA amplification products was measured using a 2100 Bioanalyzer (purchased from Agilent).

[0107] 4.cDNA library construction and sequencing

[0108] (1) Tn5 interruption

[0109] Based on the cDNA concentration, 80 ng of cDNA obtained in step 3 above was added to 0.5 μM Tn5 transposase and corresponding buffer (purchased from BGI, cat. no. 10000028493), mixed to make a 20 μL reaction system, reacted at 55°C for 10 min, and then added 5 μL of 0.1% SDS and mixed at room temperature for 5 min to complete the Tn5 shearing step.

[0110] (2) PCR amplification

[0111] Prepare 100 μL of the reaction system shown in Table 5:

[0112] Table 5: Library construction and amplification reaction system

[0113] After mixing, place the tube in a PCR instrument and set the following program: 95°C for 3 minutes, 13 cycles (98°C for 20 seconds, 58°C for 20 seconds, 72°C for 3 minutes), 72°C for 5 minutes, and 4°C for ∞. After the reaction, the DNA was purified using magnetic beads with XP beads. The dsDNA concentration was quantified using a Qubit instrument.

[0114] (3) Interrupt product double selection

[0115] The PCR amplification product and purified magnetic beads (purchased from Norvegant) were mixed at a ratio of 1:0.55. After standing for 10 minutes, the supernatant was transferred to a new PCR tube and then purified and recovered by adding 0.25× purified magnetic beads (purchased from Norvegant). The dsDNA concentration was quantified using a Qubit instrument, and the length distribution of the cDNA amplification products was measured using an Agilent 2100 Bioanalyzer.

[0116] (4) Sequencing

[0117] 80 ng of the above-mentioned double-selected products after fragmentation were taken for DNB preparation. The 40 μL reaction system was configured as shown in Table 6:

[0118] Table 6: DNB preparation system for sequencing

[0119] The reaction volume was placed in a PCR instrument under the following conditions: 95°C for 3 minutes, then 40°C for 3 minutes. After completion of the reaction, the reaction mixture was placed on ice and 40 μL of DNB preparation enzyme mix I, 2 μL of enzyme mix II, 1 μL of ATP, and 0.1 μL of T4 Ligase from the DNBSEQ sequencing kit were added. After mixing, the reaction mixture was placed in a PCR instrument at 30°C for 20 minutes to form DNBs. DNBs were loaded onto the MGISEQ2000 sequencing chip according to the instructions in the PE50 kit provided with the MGISEQ2000, and sequencing was performed according to the relevant instructions. Customized sequencing was selected, with single-strand sequencing split into two sections: a 25-bp sequence followed by a 60-cycle dark reaction and a 10-bp UMI sequence. Second-strand sequencing was set to 50 bp.

[0120] (5) Data analysis:

[0121] Log in to https: / / uat.stomics.tech / sap / login and follow the website instructions to perform data analysis.

[0122] The first 25bp of the read1 sequence obtained in PE50 sequencing (from the first-strand sequencing) is compared with the 25bp position information during the chip preparation process. The reads that can be matched to the position information on the chip are retained and mapped to the corresponding chip positions. The read2 corresponding to the reads corresponding to the chip position is found (from the second-strand sequencing), and the reads2 are compared with the human genome. The duplicate reads are removed based on the UMI information to obtain the gene situation captured in each cell and the number of reads for each gene. The captured reads are restored to the corresponding cells based on the spatial barcode. The obtained cell number is divided by the total number of cells input, and the cell capture rate of poly-lysine combined with different extracellular matrix proteins (fibronectin, vitronectin, laminin) and integrin is calculated respectively. The specific results are shown in Figure 2.

[0123] Example 2: Comparison of chips modified with polylysine alone and with proteins (fibronectin and laminin) alone and the polylysine-linked proteins (fibronectin and laminin) of the present invention

[0124] 1. Chip Modification

[0125] (1) Poly-lysine solution and fibronectin were mixed at a ratio of 1:50 and added dropwise to the surface of the chip in a 24-well plate. After rapid manual mixing, the mixture was placed on a shaker at 50 rpm and incubated at 23°C for 60 min. The experimental groups were specifically set up as follows: Sample 1 consisted of a cell suspension (2W cells) directly plated on a chip co-modified with poly-lysine and fibronectin; Sample 2 consisted of a cell suspension (2W cells) directly plated on a chip co-modified with poly-lysine and laminin; Sample 3 consisted of a cell suspension (2W cells) directly plated on a chip co-modified with poly-lysine, fibronectin, and laminin. The specific settings of the control groups are as follows: Control group 1 and control group 2 are cell suspensions (2W cells) directly plated on chips modified only with poly-lysine, control group 3 is cell suspensions (2W cells) directly plated on chips modified only with fibronectin, control group 4 is cell suspensions (2W cells) directly plated on chips modified only with laminin, and control group 5 is cell suspensions (2W cells) directly plated on chips modified only with a mixture of fibronectin and laminin.

[0126] (2) Use a pipette to remove the solution from the well plate and wash twice with 1 mL of 1× PBS.

[0127] 2. Single Cell Fixation

[0128] (1) Take approximately 20,000 PBMC cells and prepare a cell suspension (PBS solution) according to conventional methods.

[0129] (2) Drop the prepared cell suspension onto the modified chip (the probes of the chip are connected to specific DNA fragments, including Barcode, UMI, and PolyT). After incubation at room temperature for 10 minutes, place the chip in -20℃ pre-cooled methanol for 30 minutes.

[0130] (3) The cells were permeabilized by treating with hydrochloric acid and 10% pepsin for 30 seconds.

[0131] 3. cDNA Synthesis

[0132] (1) cDNA synthesis

[0133] Prepare 200 μL of the reverse transcriptase reaction system shown in Table 2 of Example 1, apply the reverse transcriptase reaction solution to the chip, cover thoroughly, and incubate at 42°C for 90-180 minutes. The reverse transcriptase will synthesize cDNA using the mRNA as a template and a polyT-containing primer, adding a CCC overhang to the 3' end of the cDNA strand. After hybridization and annealing of the TSO sequence (specifically, the sequence shown in SEQ ID NO: 1) with the cDNA strand (through complementary pairing of the GGG residue at the end of the TSO sequence with the CCC overhang of the cDNA strand), the reverse transcriptase will continue to extend the cDNA strand using the TSO sequence as a template, adding a known primer adapter to the 3' end of the cDNA strand.

[0134] (2) cDNA release

[0135] Prepare the cDNA release system as shown in Table 3 of Example 1, add 400 μL / well of cDNA Release Mix to the reaction wells of the above chip, cover the reaction wells with the chip with sealing film to seal, cover with the plate cover and seal the outer ring to prevent volatilization, and react in a 55°C incubator for 3-17 hours.

[0136] (3) cDNA amplification

[0137] Prepare 200 μL of the reaction system shown in Table 4 of Example 1, which was used for 3' end transcriptome sequencing library construction, and divided into two PCR tubes:

[0138] The above reaction system was placed in a PCR instrument and the following reaction program was set: 95°C for 3 min, 15 cycles (98°C for 20 s, 58°C for 20 s, 72°C for 3 min), 72°C for 5 min, and 4°C. After the reaction, magnetic beads (purchased from Novozymes) were purified and recovered. The dsDNA concentration was quantified using a Qubit instrument, and the length distribution of the cDNA amplification products was detected using a 2100 bioanalyzer (purchased from Agilent). The sequence of cDNA amplification primer 1 in the cDNA amplification is shown in SEQ ID NO: 2, and the sequence of cDNA amplification primer 2 is shown in SEQ ID NO: 3.

[0139] 4.cDNA library construction and sequencing

[0140] (1) Tn5 interruption

[0141] Based on the cDNA concentration, 80 ng of cDNA obtained in step 3 above was added with 0.5 μM Tn5 transposase and corresponding buffer (purchased from BGI, cat. no. 10000028493), mixed to form a 20 μL reaction system. After reacting at 55°C for 10 min, 5 μL of 0.1% SDS was added and mixed at room temperature for 5 min to complete the Tn5 shearing step.

[0142] (2) PCR amplification

[0143] Prepare 100 μL of the reaction system shown in Table 5 of Example 1, mix thoroughly, and place in a PCR instrument with the following program: 95°C for 3 min, 13 cycles (98°C for 20 s, 58°C for 20 s, 72°C for 3 min), 72°C for 5 min, and 4°C for ∞. After the reaction, magnetic bead purification and recovery were performed using XP beads. The dsDNA concentration was quantified using a Qubit instrument. The sequence of library construction primer 1 in the PCR amplification in this example is shown in SEQ ID NO: 4, and the sequence of library construction primer 2 is shown in SEQ ID NO: 5.

[0144] (3) Interrupt product double selection

[0145] The PCR amplification product and purified magnetic beads (purchased from Norvegant) were mixed at a ratio of 1:0.55. After standing for 10 minutes, the supernatant was transferred to a new PCR tube and purified with 0.25× purified magnetic beads (purchased from Norvegant). dsDNA concentration was quantified using a Qubit instrument, and the length distribution of cDNA amplification products was measured using an Agilent 2100 Bioanalyzer.

[0146] (4) Sequencing

[0147] 80 ng of the double-selected products after the above shearing were taken and DNB preparation was performed respectively, and a 40 μL reaction system as shown in Table 6 of Example 1 was prepared.

[0148] The reaction volume was placed in a PCR instrument under the following reaction conditions: 95°C for 3 min, 40°C for 3 min. After the reaction, the reaction was placed on ice, and 40 μL of the DNBSEQ sequencing kit's enzyme mix I, 2 μL of enzyme mix II, 1 μL of ATP, and 0.1 μL of T4Ligase were added. After mixing, the reaction system was placed in a PCR instrument at 30°C for 20 min to form DNBs (the primer sequence for preparing DNBs for sequencing is shown in SEQ ID NO: 6).

[0149] DNBs were loaded onto the MGISEQ 2000 sequencing chip according to the method described in the PE50 kit provided with the MGISEQ 2000. Sequencing was performed according to the relevant instructions, and customized sequencing was selected. The first strand sequencing was divided into two sections, first measuring 25 bp followed by 60 cycles of dark reaction, and then measuring the 10 bp UMI sequence. The second strand sequencing was set to measure 50 bp.

[0150] (5) Data analysis:

[0151] Log in to https: / / uat.stomics.tech / sap / login and follow the instructions for data analysis. Compare the first 25bp of the read1 sequence (from the first strand) obtained during PE50 sequencing with the 25bp position information from the chip preparation process. Retain reads that can be aligned to the chip position information and assign them to the corresponding chip position. Find read2 (from two-strand sequencing) corresponding to the reads corresponding to the chip position, align reads2 with the human genome, remove duplicate reads based on the UMI information, obtain the gene situation captured in each cell and the number of reads for each gene, restore the captured reads to the corresponding cell based on the spatial barcode, and divide the obtained cell number by the number of cells input to calculate the cell capture rate of the chip modified with polylysine alone, the chip modified with fibronectin alone, the chip modified with laminin alone, the chip modified with fibronectin and laminin alone, the chip modified with polylysine and fibronectin of the present invention, the chip modified with polylysine and laminin of the present invention, and the chip modified with polylysine, fibronectin and laminin of the present invention. See Figure 3 for specific results.

[0152] Comparative Example: Comparison of using polylysine combined with concanavalin and the polylysine combined with protein (fibronectin) modified chip of the present invention

[0153] 1. Chip Modification

[0154] (1) Mix the poly-lysine solution with concanavalin and fibronectin at a ratio of 1:50, and add them dropwise to the surface of the chip in a 24-well plate. After rapid manual mixing, place them on a shaker at 50 rpm and incubate at 23°C for 60 min.

[0155] (2) Use a pipette to remove the solution from the well plate and wash twice with 1 mL of 1× PBS.

[0156] 2. Single Cell Fixation

[0157] (1) Take approximately 20,000 PBMC cells and prepare a cell suspension (PBS solution) according to conventional methods.

[0158] (2) Drop the prepared cell suspension onto the modified chip (the probes of the chip are connected to specific DNA fragments, including Barcode, UMI, and PolyT). After incubation at room temperature for 10 minutes, place the chip in -20℃ pre-cooled methanol for 30 minutes.

[0159] 3. DAPI Staining

[0160] After the methanol evaporates and the chip surface becomes dry, use DAPI fluorescent staining solution to stain the cells on the chip at room temperature for 5 minutes. Remove the tissue fluorescent staining solution on the surface, add 0.1xSSC (RI) to wash the chip, blow dry the chip surface with a fan, add glycerol, and cover with a coverslip.

[0161] 4. Fluorescence Photography and Counting

[0162] The chip cells were photographed using the epifluorescence mode of a fluorescence microscope. After taking the pictures, the cells captured using the polylysine combined with concanavalin modified chip and the polylysine combined with fibronectin modified chip of the present invention were counted using counting software. The total number of cells obtained was divided by the total number of cells input, and finally the cell recovery rate was obtained. The specific results are shown in Figure 4.

[0163] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0164] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. Use of polylysine and protein factors in improving the cell capture efficiency of a solid support, wherein the protein factors include at least one of extracellular matrix proteins and cell integrins.

2. The use according to claim 1, characterized in that The extracellular matrix protein includes at least one of laminin, vitronectin, fibronectin, collagen, elastin, large molecular glycoprotein, adhesion protein, and cellulose.

3. The use according to claim 1, characterized in that The extracellular matrix protein includes at least one of laminin, vitronectin, and fibronectin.

4. The use according to claim 1, characterized in that The cell integrin includes at least one of integrin and adhesion protein.

5. The use according to claim 1, characterized in that The cell integrin includes integrin.

6. The use according to any one of claims 1 to 5, characterized in that The solid support includes at least one of a gel matrix, magnetic particles, a cellulose matrix, a metal ion affinity matrix, a polymer matrix, a porous material, and a gel filtration plate.

7. The use according to claim 6, characterized in that The porous material includes a microarray composed of a silicon wafer, a glass slide, or a polymer material.

8. The use according to claim 1, characterized in that The surface of the solid support includes nucleic acid molecules.

9. The use according to claim 8, characterized in that The nucleic acid molecules are labeled, and the labels include at least one of a radioactive label, a fluorescent label, an enzyme label, a biotin label, and an immunological label.

10. The use according to claim 8, characterized in that The nucleic acid molecules are modified, and the modifications include at least one of an antisense modification, a modification catalyzed by a modifying enzyme, chemical crosslinking, magnetic modification, nanopore array modification, and optical modification.

11. The use according to any one of claims 8 to 10, characterized in that The solid support is a sequencing chip.

12. A method for modifying a solid support, characterized in that Comprising: Contact-treating the solid support with a polylysine-protein factor mixture, The polylysine-protein factor mixture includes polylysine and a protein factor, The protein factor includes at least one of an extracellular matrix protein and a cell integrin.

13. The method according to claim 12, wherein the contact treatment is performed by at least one of dropwise treatment, immersion treatment, and spraying treatment of the polylysine-protein factor mixture on the solid support.

14. The method according to claim 12, characterized in that The molecular weight of the polylysine is not less than 70,000 Daltons. Preferably, the molecular weight of the polylysine is 70,000 - 300,000 Daltons. More preferably, the molecular weight of the polylysine is 70,000 - 150,000 Daltons.

15. The method according to claim 12, characterized in that In the polylysine-protein factor mixture, the mass ratio of the polylysine to the protein factor is 1:1 - 1:10,000.

16. The method according to claim 12, characterized in that In the polylysine-protein factor mixture, the mass ratio of the polylysine to the protein factor is 1:25 - 1:

50.

17. The method according to claim 12, characterized in that In the polylysine-protein factor mixture, the mass ratio of the polylysine to the protein factor is 1:1, 1:10, 1:25, 1:50, 1:100, 1:250, 1:500, 1:1000, or 1:10,000.

18. The method according to claim 12, characterized in that In the polylysine-protein factor mixture, the mass ratio of the polylysine to the protein factor is 1:25 or 1:

50.

19. The method according to any one of claims 12 to 18, characterized in that The extracellular matrix protein includes at least one of laminin, vitronectin, fibronectin, collagen, elastin, large molecular glycoprotein, adhesion protein, and cellulose.

20. The method according to any one of claims 12 to 18, characterized in that The extracellular matrix protein includes at least one of laminin, vitronectin, and fibronectin.

21. The method according to any one of claims 12 to 18, characterized in that The cell integrin includes at least one of integrin and adhesion protein.

22. The method according to any one of claims 12 to 18, characterized in that The cell integrin includes integrin.

23. The method according to any one of claims 12 to 18, characterized in that The solid support includes at least one of a gel matrix, magnetic particles, a cellulose matrix, a metal ion affinity matrix, a polymer matrix, a porous material, and a gel filtration plate.

24. The method according to claim 23, characterized in that The porous material includes a microarray composed of a silicon wafer, a glass slide, or a polymer material.

25. The method according to claim 12, characterized in that The surface of the solid support includes nucleic acid molecules.

26. The method according to claim 25, characterized in that The nucleic acid molecule is labeled, and the label includes at least one of a radioactive label, a fluorescent label, an enzyme label, a biotin label, and an immune label.

27. The method according to claim 25, characterized in that The nucleic acid molecule is modified, and the modification includes at least one of an antisense modification, a modification catalyzed by a modifying enzyme, chemical crosslinking, magnetic modification, nanopore array modification, and optical modification.

28. A solid support, characterized in that It is obtained by modification using the method according to any one of claims 12 to 27.

29. A solid support, characterized in that Comprising: A substrate layer, polylysine, and a protein factor, wherein the polylysine and the protein factor are disposed on the upper surface of the substrate layer, and the protein factor includes at least one of an extracellular matrix protein and a cell integrin.

30. The solid support according to claim 29, characterized in that The mass ratio of the polylysine to the protein factor is 1:1 to 1:10000.

31. The solid support according to claim 29, characterized in that The distribution density of the polylysine on the surface of the basal layer is 1.3x10 -10 ~5.7x10 -10 mol / cm 2 .

32. The solid support according to claim 29, characterized in that The distribution density of the protein factor on the surface of the basal layer is 1.35x10 -10 ~1.35x10 -6 mol / cm 2 .

33. A cell capture method, characterized in that Comprising: Using the method according to any one of claims 12 to 27 to perform a modification treatment on a solid support; And Using the modified solid support to perform cell capture treatment on a sample to be captured.

34. The method according to claim 33, characterized in that The sample to be captured includes at least one of a single-cell sample and a tissue section; Optionally, the single cell is provided in the form of a single-cell suspension.

35. The method according to claim 34, characterized in that When the sample to be captured is a single-cell suspension, the cell capture treatment includes: Dropping the single-cell suspension onto the surface of the modified solid support; Optionally, performing a fixation treatment on the modified solid support with the single-cell suspension dropped thereon.

36. A method for RNA capture, characterized in that, Comprising: Using the method according to any one of claims 33 to 35 to capture cells in a sample; And performing a permeabilization treatment on the captured cells to obtain RNA in the sample.

37. A library construction method, characterized in that, Comprising: Using the method according to claim 36 to obtain RNA in a sample to be tested; Performing reverse transcription treatment and amplification treatment on the RNA in the sample to be tested to obtain a sequencing library.

38. A sequencing method, characterized in that, Comprising: Using the method according to claim 37 to construct a sequencing library; And Performing a sequencing treatment on the sequencing library to obtain a sequencing result; Optionally, the sequencing treatment is performed on an MGISEQ-2000 platform.

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