Application of decrosslinking reagent in capturing nucleic acid of biological sample fixed by aldehyde reagent

Abstract

The invention provides application of a cross-linking reagent in capturing nucleic acid of a biological sample fixed by an aldehyde reagent. The decrosslinking reagent comprises a denaturing agent and a buffer solution; wherein the denaturant is selected from at least one of a compound shown as a formula (I) and salt substances thereof; the buffer solution is selected from at least one of a sodium citrate-sodium chloride buffer solution, a phosphate buffer solution, a Tris-HCl buffer solution and an HEPES buffer solution. The decrosslinking reagent can eliminate connection between protein and nucleic acid in a biological sample fixed by an aldehyde reagent, unfold a nucleic acid chain and reduce the melting temperature, so that the capture efficiency of cells and the capture number of genes are improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically, it relates to the application of decrosslinking reagents in capturing nucleic acids from biological samples immobilized with aldehyde reagents. Further, this invention relates to a method for decrosslinking biological samples immobilized with aldehyde reagents, a nucleic acid capture method, and a library construction method. Background Technology

[0002] Common decross-linking methods used in nucleic acid sequencing include the use of small molecule quenchers, such as glycine or Tris, which can react with paraformaldehyde (PFA) to prevent or reduce cross-linking. However, these methods usually require incubation at high temperatures, which often leads to cells detaching from the solid support, affecting cell capture efficiency and gene capture count.

[0003] Therefore, there is an urgent need to develop new decrosslinking methods to improve cell capture efficiency and gene capture number. Summary of the Invention

[0004] The present invention aims to at least partially address one of the technical problems existing in the prior art. To this end, the present invention provides a decrosslinking reagent for capturing nucleic acids in biological samples immobilized by aldehyde reagents.

[0005] This invention is based on the following discoveries of the inventors:

[0006] When capturing nucleic acids in immobilized biological samples, existing decrosslinking methods often result in cells detaching from solid supports (e.g., space chips), leading to low cell capture rates and poor gene capture. To overcome this problem, this invention utilizes a decrosslinking reagent in the capture of nucleic acids from aldehyde-immobilized biological samples. This decrosslinking reagent eliminates the bonds between proteins and nucleic acids in aldehyde-immobilized biological samples, unfolds the nucleic acid chains, and lowers the melting temperature, thereby improving cell capture efficiency and gene capture count, especially the capture efficiency of immobilized probes.

[0007] In a first aspect, the present invention proposes the application of a decrosslinking agent in capturing nucleic acids in biological samples immobilized by aldehyde reagents. According to an embodiment of the present invention, the decrosslinking agent comprises: a denaturing agent and a buffer; wherein the denaturing agent is selected from at least one compound of formula (I) and its salts; and the buffer is selected from at least one sodium citrate-sodium chloride buffer, phosphate buffer, Tris-HCl buffer, and HEPES buffer.

[0008]

[0009]

[0010] Where X is selected from either O or N;

[0011] R1 is selected from H, -NH2, Cl-C 10 Alkyl, C1-C 10 One of the alkoxy groups;

[0012] R2 is selected from H, -NH2, Cl-C 10 Alkyl, C1-C 10 Alkyl groups and those in the air;

[0013] Empty or direct bond. According to embodiments of the present invention, the above-mentioned decrosslinking reagent is used in the process of capturing nucleic acids of biological samples fixed with aldehyde reagents to eliminate the linkage between proteins and nucleic acids in biological samples fixed with aldehyde reagents, unfold the nucleic acid chains, reduce the melting temperature, thereby improving the capture efficiency of cells and the number of genes captured, especially the capture efficiency of fixed probes.

[0014] In a second aspect, the present invention provides a method for decrosslinking aldehyde-fixed biological samples. According to an embodiment of the present invention, the method includes: performing a decrosslinking treatment on a biological sample to be tested after fixation with an aldehyde reagent using the decrosslinking reagent described in the first aspect of the present invention. The method according to the embodiments of the present invention can improve the capture efficiency of nucleic acid-captured cells and the number of genes captured in aldehyde-fixed biological samples, especially the capture efficiency of fixed probes.

[0015] In a third aspect, the present invention provides a nucleic acid capture method. According to an embodiment of the present invention, the nucleic acid capture method includes: performing a decrosslinking treatment on a biological sample immobilized with an aldehyde reagent using the method described in the second aspect of the present invention, and permeabilizing the decrosslinking product to capture nucleic acids in the biological sample. According to the method of the embodiments of the present invention, treating the biological sample with a decrosslinking reagent before permeabilization can significantly improve the cell capture efficiency and nucleic acid capture number of nucleic acids in biological samples immobilized with aldehyde reagent, especially the capture efficiency of immobilized probes.

[0016] In a fourth aspect, the present invention provides a method for library construction. According to an embodiment of the present invention, the method for library construction includes: using nucleic acids obtained from a biological sample to be tested as a template, performed as an extension reaction using the method described in the third aspect of the present invention, and optionally performing amplification processing, to obtain a sequencing library; further, using RNA from a cell sample to be tested as a template, performed as a reverse transcription, and optionally performing amplification processing, to obtain a sequencing library. The method according to embodiments of the present invention can construct sequencing libraries with high efficiency, high fidelity, and high coverage.

[0017] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0018] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0019] Figure 1 The images show the capture results from Examples 1, 2, 1, and 2. Detailed Implementation

[0020] 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 construed as limiting the present invention.

[0021] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0022] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this invention, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] In this invention, the term "direct bond" refers to a covalent bond.

[0024] In this invention, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0025] In this invention, the terms “optionally,” “optionally,” or “optionally” generally refer to events or conditions described subsequently that may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0026] In this invention, the term "gene capture number" refers to the number of genes successfully captured and sequenced during the sequencing process.

[0027] In this invention, the minimum and maximum carbon atom content in hydrocarbon groups are indicated by prefixes. For example, the prefix Ca~b indicates the presence of "a" to "b" carbon atoms. Exemplarily, "C..." 0～n"C" refers to a saturated / unsaturated carbon chain, either straight or branched, containing 0, 1, 2, 3, 4, 5, ..., or n carbon atoms; further understanding, "C" 0～n "Should be interpreted as any subranges included, such as C" 0～6 In, containing C 0～6 C 0～3 C 0～2 C 2～6 C 2～5 C 2～4 C 2～3 C 3～6 C 3～5 C 3～4 C 4～6 C 4～5 ; "C 1～n "C" refers to a saturated / unsaturated carbon chain, either straight or branched, containing 1, 2, 3, 4, 5, ..., or n carbon atoms; further understanding, "C" 1～n "Should be interpreted as any subranges included, such as C" 1～6 In, containing C 1～6 C 1～3 C 1～2 C 2～6 C 2～5 C 2～4 C 2～3 C 3～6 C 3～5 C 3～4 C 4～6 C 4～5 The term "C1-C" 10 "Alkyl" should be understood to mean a straight-chain or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, etc.

[0028] In the chemical structure of the ligands or compounds described in this disclosure, the bonds... This indicates that the configuration is not specified. If chiral isomers exist in the chemical structure, the bond... It can be Or simultaneously include and Two configurations. Although all the above structural formulas are shown in some isomer forms for simplicity, this disclosure can include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates, and enantiomers.

[0029] This invention proposes the application of cross-linking reagents in capturing nucleic acids from biological samples fixed with aldehyde reagents, methods for decross-linking biological samples fixed with aldehyde reagents, nucleic acid capture methods, library construction methods, sequencing methods, and methods for improving cell capture efficiency in biological samples fixed with aldehyde reagents. These will be described in detail below.

[0030] Application of decrosslinking reagents in capturing nucleic acids in biological samples immobilized by aldehyde reagents

[0031] In a first aspect, the present invention proposes the application of a decrosslinking agent in capturing nucleic acids in biological samples immobilized by aldehyde reagents. According to an embodiment of the present invention, the decrosslinking agent comprises: a denaturing agent and a buffer; wherein the denaturing agent is selected from at least one compound of formula (I) and its salts; and the buffer is selected from at least one sodium citrate-sodium chloride buffer, phosphate buffer, Tris-HCl buffer, and HEPES buffer.

[0032]

[0033] Where X is selected from either O or N;

[0034] R1 is selected from H, -NH2, Cl-C 10 Alkyl, C1-C 10 One of the alkoxy groups; R2 is selected from H, -NH2, C1-C 10 Alkyl, C1-C 10 Alkyl groups and those in the air; Empty or direct bond. According to embodiments of the present invention, the above-mentioned decrosslinking reagent is used in the process of capturing nucleic acids of biological samples fixed with aldehyde reagents to eliminate the linkage between proteins and nucleic acids in biological samples fixed with aldehyde reagents, unfold the nucleic acid chains, reduce the melting temperature, thereby improving the capture efficiency of cells and the number of genes captured, especially the capture efficiency of fixed probes.

[0035] It should be noted that sodium citrate-sodium chloride buffer (SSC, sterile) is commonly used as a buffer in biochemical and molecular biology experiments. It contains sodium citrate and sodium chloride, which maintain a stable pH and provide a suitable ionic environment. In nucleic acid hybridization and elution experiments, SSC is often used to adjust the ionic strength of the solution, affecting the interactions between nucleic acid molecules. According to embodiments of the present invention, 20×SSC is used as a stock solution. 20×SSC consists of 0.3 mol / L sodium citrate and 3 mol / L sodium chloride, with a pH of 7.0, and water as the solvent. In some embodiments of the present invention, the 20×SSC stock solution is diluted to 1×SSC to 5×SSC as one of the specific components of the decrosslinking reagent described in the present invention. In an optional embodiment of the present invention, the 20×SSC stock solution is diluted to 2×SSC as one of the specific components of the decrosslinking reagent described in the present invention.

[0036] According to an embodiment of the present invention, the compound represented by formula (I) has the structure of the compound represented by formula (II):

[0037]

[0038] According to an embodiment of the present invention, when X is 0, R1 is empty, R2 is empty, and R1 is selected from H, -NH2, Cl-C. 10 Alkyl, C1-C 10 One of the alkoxy groups.

[0039] According to an embodiment of the present invention, the compound represented by formula (I) has the structure of the compound represented by formula (III):

[0040]

[0041] According to an embodiment of the present invention, when X is N, It is a direct bond, R1 is NH2, and R2 is selected from H, -NH2, Cl-C. 10 Alkyl, C1-C 10 One of the alkoxy groups.

[0042] According to an embodiment of the present invention, the compound represented by formula (I) has the following structure;

[0043]

[0044] According to an embodiment of the present invention, It is formamide.

[0045] According to an embodiment of the present invention, the salt of the compound represented by formula (I) includes at least one of the hydrochloride salt and acetate salt of the compound represented by formula (I).

[0046] According to an embodiment of the present invention, the salt of the compound shown in formula (II) includes at least one of formamide hydrochloride and formamide acetate.

[0047] According to an embodiment of the present invention, the salt of the compound represented by formula (III) includes at least one of guanidine hydrochloride, methyl guanidine hydrochloride, ethyl guanidine hydrochloride, and guanidine acetate.

[0048] According to an embodiment of the present invention, the biological sample is at least one of a cell sample and a tissue sample;

[0049] According to embodiments of the present invention, the aldehyde reagent is selected from at least one of formaldehyde, paraformaldehyde, glutaraldehyde, and glyoxal.

[0050] According to an embodiment of the present invention, the paraformaldehyde is paraformaldehyde with a weight percentage concentration of 4%.

[0051] According to an embodiment of the present invention, the solvent of the buffer solution is water, preferably nuclease-free water (NF-H2O).

[0052] According to embodiments of the present invention, the nucleic acids in the biological sample immobilized by the aldehyde-based capture reagent are captured using capture probes linked to a solid-phase support. Those skilled in the art will recognize that the nucleic acids in the biological sample immobilized by the aldehyde-based capture reagent can be used in single-cell sequencing, such as single-cell transcriptome sequencing and single-cell genome sequencing; the solid-phase support includes a chip, which includes a spatial chip.

[0053] According to embodiments of the present invention, the decrosslinking agent comprises: formamide with a volume fraction of 10% to 90%, sodium chloride with a concentration of 0.15 mol / L to 0.75 mol / L, and sodium citrate with a concentration of 0.015 mol / L to 0.075 mol / L. For example, the volume fraction of formamide can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, the concentration of sodium chloride can be 0.15 mol / L, 0.25 mol / L, 0.35 mol / L, 0.45 mol / L, 0.55 mol / L, 0.65 mol / L, or 0.75 mol / L, and the concentration of sodium citrate can be 0.015 mol / L, 0.025 mol / L, 0.035 mol / L, 0.045 mol / L, 0.055 mol / L, 0.065 mol / L, or 0.075 mol / L.

[0054] According to embodiments of the present invention, the decrosslinking agent comprises: formamide at a volume fraction of 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, or 80%–90%; sodium chloride at a concentration of 0.15 mol / L–0.3 mol / L, 0.3 mol / L–0.45 mol / L, 0.45 mol / L–0.6 mol / L, or 0.6 mol / L–0.75 mol / L; and sodium citrate at a concentration of 0.015 mol / L–0.03 mol / L, 0.03 mol / L–0.045 mol / L, 0.045 mol / L–0.06 mol / L, or 0.06 mol / L–0.075 mol / L.

[0055] Methods for decomposing biological samples fixed with cross-linking aldehyde reagents

[0056] In a second aspect, the present invention provides a method for decrosslinking aldehyde-fixed biological samples. According to an embodiment of the present invention, the method includes: performing a decrosslinking treatment on a biological sample to be tested after fixation with an aldehyde reagent using the decrosslinking reagent described in the first aspect of the present invention. The method according to the embodiments of the present invention can eliminate the linkages between proteins and nucleic acids in aldehyde-fixed biological samples, unfold the nucleic acid chains, and lower the melting temperature, thereby improving cell capture efficiency and gene capture count, especially the capture efficiency of fixed probes.

[0057] According to an embodiment of the present invention, the biological sample is at least one of a cell sample and a tissue sample.

[0058] According to embodiments of the present invention, the temperature for the decrosslinking treatment is 20°C to 99°C, for example, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 99°C, and the range between these values, 30°C to 99°C, 40°C to 99°C, 51°C to 99°C, 52°C to 99°C, and 53°C to 99°C. According to the method of the present invention, using the decrosslinking reagent described in the first aspect of the present invention for decrosslinking treatment can further unfold the nucleic acid chains, further reduce the melting temperature, thereby further improving the cell capture efficiency and gene capture number, especially the capture efficiency of immobilized probes.

[0059] According to an embodiment of the present invention, the temperature for the decrosslinking treatment is 50°C to 99°C. The method according to an embodiment of the present invention, using the decrosslinking reagent described in the first aspect of the present invention for decrosslinking treatment, can further unfold the nucleic acid chains, further reduce the decrosslinking temperature, thereby further improving the cell capture efficiency and gene capture number, especially the capture efficiency of immobilized probes.

[0060] According to an embodiment of the present invention, the temperature for the decrosslinking treatment is 50°C to 55°C. The method according to an embodiment of the present invention, using the decrosslinking reagent described in the first aspect of the present invention for decrosslinking treatment, can further unfold the nucleic acid chains, further reduce the decrosslinking temperature, thereby further improving the cell capture efficiency and gene capture number, especially the capture efficiency of immobilized probes.

[0061] According to embodiments of the present invention, the decrosslinking treatment time is 5–100 min, for example, it can be 5 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 27 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 70 min, 80 min, 90 min, and 100 min, or a range between these values, such as 5–90 min, 5–80 min, and 5–70 min. According to the method of the present invention, a suitable decrosslinking treatment time can eliminate the linkage between proteins and nucleic acids in biological samples fixed with aldehyde reagents, unfold the nucleic acid chains, and lower the melting temperature, thereby improving the cell capture efficiency and gene capture number, especially the capture efficiency of fixed probes.

[0062] According to an embodiment of the present invention, the decrosslinking treatment time is 10–100 min. According to the method of the present invention, a suitable decrosslinking treatment time can further eliminate the linkages between proteins and nucleic acids in biological samples fixed with aldehyde reagents, unfold the nucleic acid chains, and lower the melting temperature, thereby further improving the cell capture efficiency and gene capture number, especially the capture efficiency of the fixed probe.

[0063] According to an embodiment of the present invention, the decrosslinking treatment time is 10–30 min. According to the method of the present invention, a suitable decrosslinking treatment time can further eliminate the linkages between proteins and nucleic acids in biological samples fixed with aldehyde reagents, unfold the nucleic acid chains, and lower the melting temperature, thereby further improving the cell capture efficiency and gene capture number, especially the capture efficiency of the fixed probe.

[0064] According to an embodiment of the present invention, the biological sample includes at least one of a single-cell sample and a tissue section.

[0065] According to an embodiment of the present invention, the single-cell sample is provided in the form of a single-cell suspension.

[0066] According to an embodiment of the present invention, after the fixation treatment and before the decrosslinking treatment, the biological sample to be tested after the fixation treatment is further brought into contact with a solid support connected to a capture probe.

[0067] According to embodiments of the present invention, the solid support comprises a chip, which includes a spatial chip. The spatial chip contains multiple microdots, each microdot coupled to an oligonucleotide probe, which is a specific primer with a spatial barcode. In embodiments of the present invention, these microdots can be used as a probe array at specific locations for in-situ capture of DNA or mRNA at corresponding locations in cells. When cells interact with these microdots, specific DNA or mRNA binds to the corresponding probe. The captured DNA or mRNA can be further sequenced to obtain its sequence information. The method according to embodiments of the present invention can eliminate the linkage between proteins and nucleic acids in aldehyde-fixed biological samples, unfold the spatial chip probes and the transcriptome secondary structure in cells, reduce the unwinding temperature, thereby improving cell capture efficiency and gene capture number.

[0068] According to embodiments of the present invention, the chip is modified with poly-L-lysine. In some embodiments, when the biological sample to be tested is a tissue section, the chip is not modified with poly-L-lysine.

[0069] According to an embodiment of the present invention, after the decrosslinking treatment, the solid support is further cleaned using 0.1×SSC. According to an embodiment of the present invention, 20×SSC is used as a stock solution, which consists of 0.3 mol / L sodium citrate and 3 mol / L sodium chloride, with a pH of 7.0, and water as the solvent. In an optional embodiment of the present invention, the 20×SSC stock solution is diluted to 0.1×SSC as an optional cleaning agent.

[0070] Nucleic acid capture methods

[0071] In a third aspect, the present invention provides a nucleic acid capture method. According to an embodiment of the present invention, the nucleic acid capture method includes: performing a decrosslinking treatment on a biological sample immobilized with an aldehyde reagent using the method described in the second aspect of the present invention, and permeabilizing the decrosslinking product to capture nucleic acids in the biological sample. According to the method of the embodiments of the present invention, treating the biological sample with a decrosslinking reagent before permeabilization significantly improves the cell capture efficiency and nucleic acid capture count in nucleic acid capture of aldehyde-fixed biological samples, especially the capture efficiency of immobilized probes.

[0072] According to embodiments of the present invention, the nucleic acid is DNA and / or RNA; further, the nucleic acid is RNA.

[0073] According to embodiments of the present invention, after the permeabilization treatment, the permeabilization product is further subjected to pre-hybridization treatment using the decrosslinking reagent described in the first aspect of the present invention. The method according to embodiments of the present invention, performing pre-hybridization after permeabilization treatment, can further improve the cell capture efficiency and RNA or DNA capture count in nucleic acid capture in aldehyde-fixed biological samples, especially the capture efficiency of fixed probes.

[0074] According to an embodiment of the present invention, the permeation treatment is carried out under conditions of hydrochloric acid and 10% pepsin.

[0075] According to an embodiment of the present invention, after the immobilization treatment and before the decrosslinking treatment, the immobilized biological sample to be tested is further brought into contact with a solid support connected to a capture probe; the capture probe includes a capture domain that is partially or completely complementary to the target nucleic acid.

[0076] According to an embodiment of the present invention, the solid support includes a chip, and the chip includes a space chip.

[0077] As is readily understood, a spatial chip refers to a chip known to those skilled in the art for locating nucleic acids in a cellular sample. Different capture probes are fixed at different locations on the spatial chip. Each capture probe includes at least a capture domain and a localization domain. The capture domains of different capture probes may be the same or different, and the localization domains of different capture probes may be different.

[0078] According to an embodiment of the present invention, the chip has poly-L-lysine modification.

[0079] According to an embodiment of the present invention, after the decrosslinking treatment and before the permeation treatment, the solid support is further cleaned using a 0.1×SSC buffer solution.

[0080] It will be readily understood by those skilled in the art that the capture probe has a closed or free 3' end, and if an extension reaction is required to be initiated subsequently, the capture probe has a free 3' end, that is, the 3' end of the capture probe has a free hydroxyl group to initiate the extension reaction.

[0081] According to embodiments of the present invention, the RNA capture method includes: performing a decrosslinking treatment on a biological sample immobilized with an aldehyde reagent using the method described in the third aspect of the present invention, and permeabilizing the decrosslinking product to capture RNA in the sample. According to the method of the present invention, treating the biological sample with a decrosslinking reagent before permeabilization can significantly improve the cell capture efficiency and RNA capture count in nucleic acid capture of biological samples immobilized with aldehyde reagents, especially the capture efficiency of the immobilized probe.

[0082] According to embodiments of the present invention, after the permeabilization treatment, the permeabilization product is further subjected to pre-hybridization treatment using the decrosslinking reagent described in the first aspect of the present invention. The method according to embodiments of the present invention, performing pre-hybridization after permeabilization treatment, can further improve the cell capture efficiency and RNA capture number in nucleic acid capture in aldehyde-fixed biological samples, especially the capture efficiency of the fixed probe.

[0083] According to an embodiment of the present invention, the permeation treatment is carried out under conditions of hydrochloric acid and 10% pepsin.

[0084] According to an embodiment of the present invention, after the immobilization treatment and before the decrosslinking treatment, the immobilized biological sample to be tested is further brought into contact with a solid support to which a capture probe is attached; the capture probe includes a capture domain that is partially or completely complementary to the target RNA in the sample.

[0085] According to an embodiment of the present invention, the solid support includes a chip, and the chip includes a space chip.

[0086] According to an embodiment of the present invention, the chip has poly-L-lysine modification.

[0087] According to an embodiment of the present invention, after the decrosslinking treatment and before the permeation treatment, the solid support is further cleaned using a 0.1×SSC buffer solution.

[0088] It will be readily understood by those skilled in the art that the capture probe has a closed or free 3' end, and if an extension reaction is required to be initiated subsequently, the capture probe has a free 3' end, that is, the 3' end of the capture probe has a free hydroxyl group to initiate the extension reaction.

[0089] The present invention particularly recommends the following steps in the method: after the biological sample to be tested is immobilized, it is brought into contact with a solid support to which a capture probe is attached; the solid support is subjected to decrosslinking treatment, optionally cleaning treatment, permeation treatment, and optionally prehybridization treatment.

[0090] It should be noted that although a space chip is used to capture nucleic acids in the embodiments of the present invention, in reality, any modifications based on existing technology that can capture nucleic acids onto a certain carrier (especially an extracellular solid support) should be within the scope of protection of the present invention. For example, magnetic beads or gels connected with capture probes can also be used to capture nucleic acids. Furthermore, the capture method is not limited to hybridization or complementation; covalent linkage can also be used to capture nucleic acids. For example, nucleic acids can be extended using primers labeled with reactive group A to obtain labeled nucleic acids, which are then captured by a support (such as a chip, gel, or magnetic bead) containing reactive group B through their linked reactive group A. Under permissible reaction conditions, reactive group A and reactive group B react to form a covalent link.

[0091] Methods for building a library

[0092] In a fourth aspect, the present invention provides a method for library construction. According to an embodiment of the present invention, the method for library construction includes: using nucleic acids obtained from a biological sample to be tested as a template, performed as an extension reaction using the method described in the third aspect of the present invention, and optionally performing amplification processing, to obtain a sequencing library; further, using RNA from a cell sample to be tested as a template, performed as a reverse transcription process, and optionally performing amplification processing, to obtain a sequencing library. The method according to the embodiments of the present invention can construct sequencing libraries with high efficiency, high fidelity, and high coverage.

[0093] According to an embodiment of the present invention, the biological sample includes at least one of a cell sample and a tissue sample.

[0094] According to an embodiment of the present invention, the biological sample includes at least one of a single-cell sample and a tissue section.

[0095] According to an embodiment of the present invention, the extension reaction or reverse transcription is initiated using the capture probe as a primer.

[0096] According to an embodiment of the present invention, the extension reaction is carried out on a solid support.

[0097] sequencing methods

[0098] In a fifth aspect, the present invention provides a sequencing method. According to an embodiment of the present invention, the sequencing method includes: constructing a sequencing library using the method described in the fourth aspect of the present invention; and performing sequencing processing on the sequencing library to obtain sequencing results. The method according to an embodiment of the present invention can obtain high-resolution sequencing data.

[0099] According to an embodiment of the present invention, the sequencing process is performed on the MGISEQ-2000 platform.

[0100] Methods to improve cell capture efficiency in biological samples fixed with aldehyde reagents

[0101] In a sixth aspect, the present invention provides a method for improving cell capture efficiency in aldehyde-fixed biological samples. According to an embodiment of the present invention, the method includes: performing a decrosslinking treatment on a biological sample to be tested after fixation with an aldehyde reagent using the decrosslinking reagent described in the first aspect of the present invention. The method according to the embodiment of the present invention can improve cell capture efficiency in aldehyde-fixed biological samples.

[0102] According to an embodiment of the present invention, the temperature for the decrosslinking treatment is 20°C to 99°C. The method according to an embodiment of the present invention, using the decrosslinking reagent described in the first aspect of the present invention for decrosslinking treatment, can unfold the nucleic acid chains, lower the melting temperature, and thereby improve the cell capture efficiency in biological samples fixed with aldehyde reagents.

[0103] According to an embodiment of the present invention, the temperature for the decrosslinking treatment is 50°C to 99°C. The method according to an embodiment of the present invention, using the decrosslinking reagent described in the first aspect of the present invention for decrosslinking treatment, can further unfold the nucleic acid chains, further reduce the decrosslinking temperature, and thereby further improve the cell capture efficiency in biological samples fixed with aldehyde reagents.

[0104] According to an embodiment of the present invention, the temperature for the decrosslinking treatment is 50°C to 55°C. The method according to an embodiment of the present invention, using the decrosslinking reagent described in the first aspect of the present invention for decrosslinking treatment, can further unfold the nucleic acid chains, further reduce the decrosslinking temperature, and thereby further improve the cell capture efficiency in biological samples fixed with aldehyde reagents.

[0105] According to an embodiment of the present invention, the decrosslinking treatment time is 5–100 min. According to the method of the present invention, a suitable decrosslinking treatment time can eliminate the linkages between proteins and nucleic acids in aldehyde-fixed biological samples, unfold the nucleic acid chains, and lower the melting temperature, thereby improving the cell capture efficiency in aldehyde-fixed biological samples.

[0106] According to an embodiment of the present invention, the decrosslinking treatment time is 10–100 min. According to the method of the present invention, a suitable decrosslinking treatment time can further eliminate the linkages between proteins and nucleic acids in aldehyde-fixed biological samples, further unfold the nucleic acid chains, lower the melting temperature, and thereby further improve the cell capture efficiency in aldehyde-fixed biological samples.

[0107] According to an embodiment of the present invention, the decrosslinking treatment time is 10–30 min. According to the method of the present invention, a suitable decrosslinking treatment time can further eliminate the linkages between proteins and nucleic acids in aldehyde-fixed biological samples, further unfold the nucleic acid chains, lower the melting temperature, and thereby further improve the cell capture efficiency in aldehyde-fixed biological samples.

[0108] According to embodiments of the present invention, the aldehyde reagent is selected from at least one of formaldehyde, paraformaldehyde, glutaraldehyde, and glyoxal.

[0109] According to an embodiment of the present invention, the biological sample to be tested includes at least one of a single-cell sample and a tissue section.

[0110] According to an embodiment of the present invention, the single-cell sample is provided in the form of a single-cell suspension.

[0111] According to an embodiment of the present invention, after the fixation treatment and before the decrosslinking treatment, the biological sample to be tested after the fixation treatment is further brought into contact with a solid support connected to a capture probe.

[0112] According to an embodiment of the present invention, the solid support includes a chip, and the chip includes a space chip.

[0113] According to an embodiment of the present invention, the chip has poly-L-lysine modification.

[0114] According to an embodiment of the present invention, after the decrosslinking treatment, the solid support is further cleaned using a 0.1×SSC buffer solution.

[0115] After the cleaning process, the decrosslinking product was permeated with hydrochloric acid and 10% pepsin.

[0116] After the permeation treatment, the permeation product is pre-hybridized using the decrosslinking reagent described in the first aspect of the present invention.

[0117] The sequence information involved in this embodiment is shown in the table below:

[0118] Table 1: Sequence Information

[0119]

[0120] Note: "r" indicates that the nucleotide at its 3' adjacent position is a ribonucleotide; "+" indicates that the nucleotide at its 3' adjacent position is modified with LNA (locked nucleotide); "*" indicates thiophosphate modification; "p" indicates phosphorylation modification; N = A, T, Cor G; V = A, Cor G.

[0121] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0122] Example 1:

[0123] I. Poly-Lysine Processing Space Chip

[0124] 1. Take 400 μL of poly-L-lysine (PLL) solution and drop it onto the surface of the space chip (BGI, Stereo-seq chip T (1cm*1cm) catalog number: 100CT112) in a 24-well plate. Shake quickly by hand until well mixed, then place on a shaker and shake at 50 rpm for 30 min at 23°C.

[0125] 2. Use a pipette tip to remove the PLL from the well plate, wash twice with 1 mL of nuclease-free water (NF-H2O), and then blow dry with a gas cylinder.

[0126] II. Single-cell fixation, decrosslinking, and permeabilization

[0127] 1. After thawing and freezing peripheral blood mononuclear cells (PBMCs), centrifuge to remove the cell suspension supernatant. Add 200 μL of 4% paraformaldehyde to the cell pellet, fix at room temperature for 10 min, then add 1 mL of PBS, mix well, and centrifuge to remove the supernatant. Repeat the PBS washing once, add an appropriate amount of PBS to disperse the cells, count the cells, and take 20,000 cells / cell array.

[0128] 2. Drop the prepared cell suspension onto the poly-L-lysine-treated space chip (20,000 cells / space chip), incubate at room temperature for 10 minutes, and then bake the space chip in a 37°C oven to dry.

[0129] 3. Prepare the decrosslinking reagent: formamide (40 μL), 20×SSC (20 μL), NF-H2O (140 μL). Immerse the cell-coated space chip in the decrosslinking reagent, incubate at 55°C for 20 min, and then wash the space chip once with 0.1×SSC.

[0130] 4. Treat the cells with hydrochloric acid and 10% pepsin for 30 seconds to permeate them.

[0131] III. cDNA Synthesis

[0132] 1. cDNA synthesis

[0133] Prepare a 200 μL reverse transcriptase reaction system as shown in Table 2. Add the reaction solution to the space chip, ensuring complete coverage, and incubate at 42℃ for 90-180 min. The reverse transcriptase will use mRNA as a template and primers containing polyT to synthesize cDNA, adding a CCC overhang to the 3' end of the cDNA strand. After hybridization and annealing of the targeted sequencing oligonucleotide (TSO) sequence with the cDNA strand (through complementary pairing of the GGG at the TSO sequence end with the CCC overhang of the cDNA strand), the reverse transcriptase will continue to extend the cDNA strand using the TSO as a template, attaching a known primer adapter to the 3' end of the cDNA. The reverse transcription reaction system can use reagents from the Stereo-seq Transcriptomics Kit T (catalog number: 101KT114).

[0134] Table 2: Reverse Transcription Reaction System

[0135]

[0136] (2) cDNA release

[0137] Configure the cDNA release system as shown in Table 3.

[0138] Table 3: cDNA release system

[0139]

[0140] Add 400 μL / well of cDNA release mix to the reaction wells of the aforementioned space chip, seal the reaction wells containing the space chip with sealing film, cover with the plate cap and seal the outer ring to prevent volatilization, and react in an incubator at 55°C for 3-17 hours.

[0141] (3) cDNA amplification

[0142] Prepare 200 μL of the reaction system shown in Table 4, and divide it into two tubes for PCR, one for 3' transcriptome sequencing library construction.

[0143] Table 4: cDNA amplification system

[0144]

[0145]

[0146] The above reaction system was placed in a PCR instrument and the following reaction program was set: 95℃ for 3 min, 15 cycles (98℃ for 20 s, 58℃ for 20 s, 72℃ for 3 min), 72℃ for 5 min, 4℃∞. After the reaction, the magnetic beads (purchased from Novizan) were purified and recovered. The concentration of dsDNA was quantified using a Qubit instrument, and the length distribution of cDNA amplification products was detected using a 2100 bioanalyzer (purchased from Agilent).

[0147] IV. cDNA Library Construction and Sequencing

[0148] (1) Tn5 interrupts

[0149] Based on the cDNA concentration, take 80 ng of cDNA obtained in step 3 above, add 0.5 μM Tn5 transposase and corresponding buffer (purchased from BGI, catalog number 10000028493), mix well to prepare a 20 μL reaction system, react at 55℃ for 10 min, add 5 μL of 0.1% SDS, mix well at room temperature for 5 min to end the Tn5 interruption step.

[0150] (2) PCR amplification

[0151] Prepare a 100 μL reaction system as shown in Table 5:

[0152] Table 5: Library construction and amplification reaction system

[0153]

[0154] After mixing, the mixture was placed in a PCR instrument and programmed as follows: 95℃ for 3 min, 13 cycles (98℃ for 20 s, 58℃ for 20 s, 72℃ for 3 min), 72℃ for 5 min, 4℃ to infinity. After the reaction, the DNA was purified and recovered using magnetic cross-linked beads (XP beads). The concentration of dsDNA was quantified using a Qubit instrument.

[0155] (3) Interrupting product selection

[0156] The PCR amplification products and purified magnetic beads (purchased from Novizan) were mixed at a ratio of 1:0.55. After standing for 10 min, the supernatant was transferred to a new PCR tube, and 0.25× purified magnetic beads (purchased from Novizan) were added for magnetic bead purification and recovery. The concentration of dsDNA was quantified using a Qubit instrument, and the length distribution of cDNA amplification products was detected using a 2100 bioanalyzer (purchased from Agilent).

[0157] (4) Sequencing

[0158] Take 80 ng of the above-mentioned interrupted double-selected product for DNB preparation. Prepare 40 μL reaction systems as shown in Table 6:

[0159] Table 6: DNB preparation system for sequencing

[0160]

[0161] Place the above reaction volume in a PCR instrument and react under the following conditions: 95℃ for 3 min, 40℃ for 3 min. After the reaction, place on ice and add 40 μL of mixed enzyme I, 2 μL of mixed enzyme II, 1 μL of adenosine triphosphate (ATP), and 0.1 μL of T4 ligase from the DNBSEQ sequencing kit for DNB preparation. Mix well and place the reaction system in the PCR instrument at 30℃ for 20 min to form DNB. Following the instructions in the PE50 kit accompanying the MGISEQ 2000, load the DNB onto the MGISEQ 2000 sequencing chip and perform sequencing according to the relevant instructions. Select customized sequencing, where the first strand sequencing is divided into two segments: first, 25 bp is sequenced, followed by 60 cycles of dark reaction, and then a 10 bp UMI sequence is sequenced. The second strand sequencing is set to 50 bp.

[0162] Example 2

[0163] Comparative Example 1 is basically the same as Example 1, except that after permeation, a pre-hybridization treatment is performed. A mixture of 40 μL formamide, 20 μL 20×SSC, and 140 μL NF-H2O is added to the space chip as a pre-hybridization solution and incubated in an oven at 55°C for 5 min. Other contents are the same as in Example 1.

[0164] Comparative Example 1

[0165] Comparative Example 1 is basically the same as Example 2, except that Comparative Example 1 involves a non-repeating crosslinking step, while the other contents are the same as Example 1.

[0166] Comparative Example 2

[0167] Comparative Example 2 is basically the same as Example 2, except that it uses a Tris EDTA buffer solution with a pH of 9 at 55°C to decrosslink for 20 min. Other contents are the same as in Example 2.

[0168] The sequencing data obtained from Examples 1-2 and Comparative Examples 1-2 were analyzed, and the data was logged into the website.

[0169] At https: / / uat.stomics.tech / sap / login, follow the website's instructions for data analysis. The first 25bp of the read1 sequence obtained from PE50 sequencing (from single-strand sequencing) is compared with the 25bp position information from the spatial chip preparation process. Reads that can be matched to the spatial chip position information are retained and mapped to their corresponding spatial chip positions. Find the read2 (from two-strand sequencing) corresponding to the reads at the spatial chip positions, compare read2 with the human genome, and remove duplicate reads based on UMI information to obtain the captured genes in each cell and the number of reads for each gene. Use the spatial barcode to reconstruct the captured reads onto the corresponding cells. The resulting cell count is divided by the total number of cells input to calculate the cell capture rates for Examples 1-2 and Comparative Examples 1-2, respectively.

[0170] The results are as follows Figure 1 As shown (corresponding to Examples 1-2 and Comparative Examples 1-2 respectively), the results show that in Example 1, the number of genes captured for sequencing after capture was 753, with a capture efficiency of 34%; in Example 2, the number of genes captured for sequencing was 604, with a capture efficiency of 71%; in Comparative Example 1, the number of genes captured for sequencing after capture was extremely low, the capture efficiency was low, and a large area of ​​cells was lost on the space chip; in Comparative Example 2, the number of genes captured for sequencing after capture was 333, the capture efficiency was 3%, and a large area of ​​cells was lost on the space chip.

[0171] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0172] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The application of a decrosslinking agent in capturing nucleic acids in biological samples fixed with aldehyde reagents, wherein the decrosslinking agent comprises: A denaturing agent and a buffer solution; wherein the denaturing agent is selected from at least one of the compounds represented by formula (I) and their salts; and the buffer solution is selected from at least one of sodium citrate-sodium chloride buffer, phosphate buffer, Tris-HCl buffer, and HEPES buffer. Where X is selected from either O or N; R1 is selected from H, -NH2, Cl-C 10 Alkyl, C1-C 10 One of the alkoxy groups; R2 is selected from H, -NH2, Cl-C 10 Alkyl, C1-C 10 Alkyl groups and those in the air; Empty or direct key.

2. The application according to claim 1, characterized in that, The compound shown in formula (I) has the structure of the compound shown in formula (II): Optionally, the compound represented by formula (I) has the structure of the compound represented by formula (III): Optionally, the compound represented by formula (I) has the following structure; Optionally, the salts of the compounds represented by formula (I) include at least one of the hydrochloride salts and acetate salts of the compounds represented by formula (I); Optionally, the salts of the compounds shown in formula (II) include at least one of formamide hydrochloride and formamide acetate; Optionally, the salt of the compound represented by formula (III) includes at least one of guanidine hydrochloride, methyl guanidine hydrochloride, ethyl guanidine hydrochloride, and guanidine acetate; Optionally, the biological sample is at least one of cell samples and tissue samples; Optionally, the aldehyde reagent is selected from at least one of formaldehyde, paraformaldehyde, glutaraldehyde, and glyoxal; Optionally, the solvent of the buffer solution is water, preferably nuclease-free water; Optionally, the nucleic acids in the biological sample immobilized by the aldehyde-capturing reagent are captured via capture probes linked to a solid support.

3. The application according to claim 1 or 2, characterized in that, include: Formamide with a volume fraction of 10%–90%, sodium chloride with a concentration of 0.15 mol / L–0.75 mol / L, and sodium citrate with a concentration of 0.015 mol / L–0.075 mol / L; Optionally, the decrosslinking agent comprises: formamide at a volume fraction of 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, or 80%–90%; sodium chloride at a concentration of 0.15 mol / L–0.3 mol / L, 0.3 mol / L–0.45 mol / L, 0.45 mol / L–0.6 mol / L, or 0.6 mol / L–0.75 mol / L; and sodium citrate at a concentration of 0.015 mol / L–0.03 mol / L, 0.03 mol / L–0.045 mol / L, 0.045 mol / L–0.06 mol / L, or 0.06 mol / L–0.075 mol / L.

4. A method for de-crosslinking aldehyde-fixed biological samples, characterized in that, include: The cross-linking agent according to any one of claims 1 to 3 is used to perform cross-linking treatment on the biological sample to be tested after fixation with aldehyde reagent; Optionally, the biological sample is at least one of a cell sample and a tissue sample.

5. The method according to claim 4, characterized in that, The temperature for the decrosslinking treatment is 20℃~99℃, preferably 50℃~99℃, and more preferably 50℃~55℃; Optionally, the decrosslinking treatment time is 5 to 100 min, preferably 10 to 100 min, and more preferably 10 to 30 min; Optionally, the biological sample includes at least one of single-cell samples and tissue sections; Optionally, the single-cell sample is provided in the form of a single-cell suspension.

6. A method for capturing nucleic acids, characterized in that, include: The method described in claim 4 or 5 is used to decrosslink the biological sample to be tested after it has been immobilized with an aldehyde reagent, and the decrosslinking product is permeabilized in order to capture the nucleic acid in the biological sample to be tested. Optionally, the nucleic acid is DNA and / or RNA; further, the nucleic acid is RNA.

7. The method according to claim 6, characterized in that, Following the permeation treatment, the permeation product is further subjected to prehybridization treatment using the decrosslinking agent described in any one of claims 1 to 3.

8. The method according to claim 6 or 7, characterized in that, The permeation treatment was carried out under conditions of hydrochloric acid and 10% pepsin.

9. The method according to claim 6 or 7, characterized in that, After the immobilization process and before the decrosslinking process, the immobilized biological sample to be tested is further brought into contact with a solid support with a capture probe attached; the capture probe includes a capture domain that is partially or completely complementary to the target nucleic acid in the sample. Optionally, the solid support includes a chip, and the chip includes a space chip; Optionally, the chip is modified with poly-L-lysine; Optionally, after the decrosslinking treatment and before the permeation treatment, the solid support is further cleaned using a 0.1×SSC buffer.

10. A method for constructing a library, characterized in that, include: Using the nucleic acid in the biological sample to be tested obtained by the method according to any one of claims 6 to 9 as a template, an extension reaction is performed, and optional amplification treatment is carried out to obtain a sequencing library; Furthermore, reverse transcription is performed on the RNA in the cell sample to be tested obtained by the method according to any one of claims 6 to 9, and optional amplification processing is performed to obtain a sequencing library; Optionally, the biological sample includes at least one of cell samples and tissue samples; Optionally, the extension reaction or reverse transcription is initiated using the capture probe as a primer; Preferably, the extension reaction takes place on a solid support.

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