Analysis method for m7G modification in single cell

Abstract

The invention particularly relates to an analysis method for m7G modification in a single cell, a method for obtaining RNA with modification, and a library for analyzing modification information carried by the RNA. The invention also relates to a method for simultaneously analyzing the transcriptome RNA and the RNA with the modification contained in the transcriptome RNA.

Description

Technical Field

[0001] The present application relates to the field of sequencing, and more particularly to a method for obtaining RNA with modifications, and a library for analyzing modification information carried by RNA. The present application also relates to a method for simultaneously analyzing transcriptome RNA and the modified RNA contained therein. Background Art

[0002] 7-Methylguanine (N 7 -methylguanosine, m 7 G) modification is one of the common forms of post-transcriptional nucleic acid base modification. Studies have shown that the guanosine N 7 In recent years, researchers have also found that mRNA internal regions also have m 7 G modification by modifying the internal mRNA in human and mouse cell lines 7 The G modification level was detected and it was found that about 0.02%-0.05% of G was methylated, which is about RNA m 6 In addition, some researchers have found that after knocking down METTL1 in HeLa cells, more than 75% of the mRNA internal m 7 The modification level of G-modified sites decreased significantly, indicating that METTL1 is not only a 7 In addition to G-modifying methyltransferases, it also regulates mRNA internal m 7 G modification, and mRNA internal m 7 G modification can promote mRNA translation. Another study found that the internal m 7 G modification is enriched in the 5' untranslated region (5'UTR), especially in the AG-rich region near the translation start site, and under the treatment of H2O2 and heat stimulation, m 7 G stress up-regulated modifications, with significant increases in modifications in the coding region (CDS) and 3' untranslated region (3'UTR) and promoted m 7 G modified mRNA translation efficiency.

[0003] RNAm 7 G-modified sequencing technologies mainly include single nucleotide resolution m- 7 G tRNA reduction and fragmentation sequencing (m 7 G tRNAReduction and Cleavage-Sequence, TRAC-Seq) technology and m 7 G-Seq, and m-based 7 G Antibody high-throughput sequencing technology, including:7 G methylated RNA co-precipitation sequencing (m 7 GMethylated RNAImmunoprecipitation-Sequence, m 7 G MeRIP-Seq) and m 7 G single-base resolution UV cross-linking co-precipitation sequencing (m 7 G individual-nucleotide-resolution cross-linking and immunoprecipitation, m 7 G miCLIP-Seq).

[0004] These technologies have opened up new avenues for RNAm 7 However, these techniques rely on large or regular amounts of RNA. For rare cells or single-cell samples such as eggs and embryos, RNAm cannot be analyzed using existing techniques due to the extremely low amount of RNA. 7 G modification detection.

[0005] Therefore, the development of single-cell RNA 7 Single cell mG sequencing technology 7 G-seq: sc-m 7 G-seq) to help map m 7 G modification map. In addition, it is helpful for studying the heterogeneity of RNA epigenetic modifications at the single-cell level in processes such as early embryonic development, oogenesis, and tumorigenesis and metastasis. Summary of the invention

[0006] The inventors of the present application provide two uniquely designed methods for labeling RNA. The first method is suitable for single cells or low-throughput single cells (less than 50), i.e., low-throughput single-cell samples; the second method is suitable for a large number of cells (50 to 10 6 Furthermore, the inventors of the present application used immunoprecipitation to capture m 7 G-modified RNA, and sequenced and analyzed it to obtain m 7 G modified information.

[0007] In addition, the inventors of the present application also found that in the labeled RNA, the capture of m 7 After the RNA modified with mG, the remaining RNA sample also has key information. 7G-modified RNA samples were separately library-established and sequenced, and after integrating the sequencing data, it was found that it could reflect the transcriptome information. Therefore, the method of the present application can achieve simultaneous extraction of RNA samples with m 7 G modified RNA sample information analysis and transcriptome analysis.

[0008] A method for labeling RNA for high-throughput single-cell samples

[0009] In a first aspect, the present application provides a method for labeling RNA of a single cell, the method comprising:

[0010] (I) providing a sample containing cells, and fixing and / or permeabilizing the cells;

[0011] (II) fragmenting the intracellular RNA to obtain multiple RNA fragments;

[0012] (III) labeling the RNA fragments, the specific steps are as follows:

[0013] (a) providing a first linker set, wherein the first linker set comprises at least one first chain and a second chain,

[0014] The first strand comprises a linker sequence and a template sequence, wherein the linker sequence is a random arrangement of 6 to 12 (e.g., 6, 7, 8, 9, 10, 11, 12) bases, and each first strand contains a different linker sequence and the same template sequence; the second strand comprises a first template sequence and a second template sequence, and the first template sequence of the second strand is complementary to the template sequence of the first strand,

[0015] Optionally, adenylase is contacted with the first strand, and the first linker group is contacted with the RNA fragment under conditions that allow nucleic acid hybridization or annealing, so that the first strand of the first linker group is connected to the 3' end of the RNA fragment;

[0016] (b) providing a second linker set, wherein the second linker set comprises at least one third chain,

[0017] The third strand comprises a first template sequence, a linker sequence and a second template sequence, the linker sequence is a random arrangement of 6 to 12 (e.g., 6, 7, 8, 9, 10, 11, 12) bases, and each third strand contains a different linker sequence and the same first template sequence and second template sequence; the first template sequence of the third strand is complementary to the second template sequence of the second strand in the first linker group,

[0018] contacting the second linker set with the product of step (a) under conditions that allow nucleic acid hybridization or annealing, so that the second linker set is ligated to the 3' end of the first strand of the first linker set;

[0019] Optionally, contacting a first neutralizing primer (e.g., the second strand) with the product obtained after the above annealing under conditions that allow nucleic acid hybridization or annealing, wherein the first neutralizing primer (e.g., the second strand) is complementary to the first template sequence, the second template sequence, or a partial sequence thereof of the third strand;

[0020] (c) providing a third linker set, wherein the third linker set comprises at least one fourth chain and a fifth chain,

[0021] The fourth strand comprises a first template sequence, a linker sequence and a second template sequence, the linker sequence is a random arrangement of 6 to 12 (e.g., 6, 7, 8, 9, 10, 11, 12) bases, and each fourth strand contains a different linker sequence and the same first template sequence and second template sequence,

[0022] The fifth strand comprises a first template sequence and a second template sequence, wherein the first template sequence of the fifth strand is complementary to the second template sequence of the third strand, and the second template sequence of the fifth strand is complementary to the first template sequence of the fourth strand,

[0023] contacting the third linker set with the product of step (b) under conditions that allow nucleic acid hybridization or annealing, so that the fourth strand of the third linker set is ligated to the 3' end of the second linker set;

[0024] Optionally, under conditions that allow nucleic acid hybridization or annealing, contacting a second neutralizing primer (e.g., the fifth strand) with the product obtained after the above annealing, wherein the second neutralizing primer (e.g., the fifth strand) is complementary to the first template sequence, the second template sequence, or a partial sequence thereof of the fourth strand in the third adapter set;

[0025] (d) providing a fourth linker set, wherein the fourth linker set comprises at least one sixth chain and a seventh chain,

[0026] The sixth strand comprises a first template sequence, a linker sequence and a second template sequence, the linker sequence is a random arrangement of 14 to 20 (e.g., 14, 15, 16, 17, 18, 19, 20) bases, and each sixth strand contains a different linker sequence and the same first template sequence and second template sequence,

[0027] The seventh strand comprises a first template sequence and a second template sequence, wherein the first template sequence of the seventh strand is complementary to the second template sequence of the fourth strand, and the second template sequence of the seventh strand is complementary to the first template sequence of the sixth strand,

[0028] contacting the fourth linker set with the product of step (c) under conditions that allow nucleic acid hybridization or annealing, so that the sixth strand of the fourth linker set is linked to the 3' end of the fourth strand of the third linker set;

[0029] Optionally, under conditions allowing nucleic acid hybridization or annealing, contacting a third neutralizing primer (e.g., the seventh strand) with the product obtained after the above annealing, wherein the third neutralizing primer (e.g., the seventh strand) is complementary to the first template sequence, the second template sequence, or a partial sequence thereof of the sixth strand in the fourth adapter set;

[0030] (e) providing a fifth linker set, wherein the fifth linker set comprises at least one eighth chain,

[0031] The eighth strand comprises a first template sequence, a linker sequence and a second template sequence, the linker sequence is a random arrangement of 6 to 12 (e.g., 6, 7, 8, 9, 10, 11, 12) bases, and each eighth strand contains a different linker sequence and the same first template sequence and second template sequence; the first template sequence of the eighth strand is complementary to the second template sequence of the sixth strand,

[0032] contacting the fifth linker set with the product of step (d) under conditions that allow nucleic acid hybridization or annealing, so that the fifth linker set is linked to the 3' end of the sixth strand of the fourth linker set;

[0033] (f) contacting a DNA polymerase with the product obtained in step (g) under conditions that allow nucleic acid ligation;

[0034] (IV) sorting single cells to obtain RNA-labeled single cells; optionally, lysing the single cells to obtain labeled RNA.

[0035] In a second aspect, the present application provides a method for obtaining a modified RNA, wherein the method further comprises, after steps (I) to (IV) as described above:

[0036] (V) providing an antibody that can specifically bind to the modified RNA, and contacting the antibody with the modified RNA; for example, providing an antibody that can specifically bind to the modified RNA 7 G modified RNA antibody, so that the antibody with m 7 G modified RNA contacts.

[0037] In certain embodiments, the modified RNA is selected from: 7 G modified RNA with m 5 C-modified RNA with m 6A modified RNA, or any combination thereof.

[0038] In certain embodiments, after step (V), the remaining RNA is collected.

[0039] In certain embodiments, prior to step (V), the labeled RNA obtained in step (IV) is purified.

[0040] In certain embodiments, the sequence of the first chain, the third chain, the fourth chain or the sixth chain of each round of the linker group contains a specific 6-12 base sequence, which is called a linker sequence. The principle of linker sequence design is specificity. In certain embodiments, the linker sequence is any permutation and combination of 8 bases. In certain embodiments, the sequence of the linker sequence will not be the same if the bases are changed by 2 or less. This is to prevent errors caused by sequencing errors. In certain embodiments, the number of linker sequences (i.e., the first chain, the third chain, the fourth chain or the sixth chain containing the linker sequence) corresponds to the number of single cells in the experiment, and the number of linker sequences (i.e., the first chain, the third chain, the fourth chain or the sixth chain containing the linker sequence) must be greater than the number of cells detected by one sequencing. The larger the number, the better, but the corresponding operation workload also increases, so the number is generally 10 times the number of cells.

[0041] In certain embodiments, the sequences of the first chain, the second chain, the third chain, the fourth chain, the fifth chain, the sixth chain, the seventh chain, and the eighth chain in each round of the joint group all contain template sequences in order to be complementary to the remaining chains. For example, in certain embodiments, the first template sequence of the second chain is complementary to the template sequence of the first chain. In such embodiments, as long as the first template sequence and the template sequence that can achieve the above-mentioned complementary function are within the scope of the present application, they are not limited to the sequences specifically used in the embodiments. The method of designing the first template sequence and the template sequence that can complement each other is known to those skilled in the art.

[0042] In certain embodiments, the first strand in the first linker set undergoes adenylylation reaction. In certain embodiments, the first strand in the first linker set undergoes adenylylation reaction by adenylase.

[0043] In certain embodiments, the first strand, the third strand, the fourth strand, and the sixth strand are all phosphorylated. In certain embodiments, the 5'

[0044] Both ends are phosphorylated.

[0045] In certain embodiments, the second template sequence of the sixth strand is complementary to a primer used for sequencing.

[0046] In certain embodiments, in step (II), the intracellular RNA is fragmented, the 5' end cap of the RNA is removed, and then the 3' end of the RNA fragment is repaired to a hydroxyl group.

[0047] In certain embodiments, the RNA shearing method is selected from ion and high temperature heating methods or RNA enzymatic cleavage methods;

[0048] In certain embodiments, the 3' end repair is performed by contacting the broken RNA with a nucleic acid polymerase (eg, T4 PNK enzyme).

[0049] In certain embodiments, in step (IV), single cells are sorted by flow cytometry.

[0050] In certain embodiments, the sample comprises a plurality of cells, for example, 50 to 10 2 cells, 10 2 to 10 3 cells, 10 3 to 10 4 cells, 10 5 to 10 6 cells.

[0051] In certain embodiments, the sample is a sample obtained from a non-mammal (eg, a zebrafish embryo), or a sample obtained from a mammal (eg, a mouse, a human).

[0052] In certain embodiments, in steps (b) to (d), a DNA polymerase (eg, a DNA ligase, eg, T4 DNA ligase) is provided.

[0053] In certain embodiments, the RNA comprises mRNA, tRNA, rRNA, or any combination thereof.

[0054] In certain embodiments, the RNA comprises mRNA and / or tRNA.

[0055] In certain embodiments, in step (a), the linking sequence is a random arrangement of 9 bases.

[0056] In certain embodiments, in steps (b) and (c), the linking sequence is a random arrangement of 8 bases.

[0057] In certain embodiments, in step (d), the linker sequence is a random arrangement of 16 bases.

[0058] In certain embodiments, in step (e), the linker sequence is a random arrangement of 8 bases.

[0059] In certain embodiments, the length of the first strand template sequence is 16-22 bases (eg, 19 bases).

[0060] In certain embodiments, the first template sequence of the second strand has a length of 16-22 bases (eg, 19 bases).

[0061] In certain embodiments, the second template sequence of the second strand has a length of 13-18 bases (eg, 15 bases).

[0062] In certain embodiments, the length of the first template sequence of the third strand is 13-18 bases (eg, 15 bases).

[0063] In certain embodiments, the second template sequence of the third strand has a length of 13-18 bases (eg, 15 bases).

[0064] In certain embodiments, the first template sequence of the fourth strand has a length of 13-18 bases (eg, 15 bases).

[0065] In certain embodiments, the second template sequence of the fourth strand has a length of 13-18 bases (eg, 15 bases).

[0066] In certain embodiments, the length of the first template sequence of the fifth strand is 13-18 bases (eg, 15 bases).

[0067] In certain embodiments, the second template sequence of the fifth strand has a length of 13-18 bases (eg, 15 bases).

[0068] In certain embodiments, the length of the first template sequence of the sixth strand is 13-18 bases (eg, 15 bases).

[0069] In certain embodiments, the length of the second template sequence of the seventh strand is 18-22 bases (eg, 20 bases).

[0070] In certain embodiments, the length of the first template sequence of the eighth strand is 20-24 bases (eg, 22 bases).

[0071] In certain embodiments, the second template sequence of the eighth strand has a length of 30-36 bases (eg, 33 bases).

[0072] In certain embodiments, the first linker set comprises 2 to 30, 30 to 60, 60 to 90, 90 to 120, 120 to 150, 150 to 180, or 180 to 210 first strands.

[0073] In certain embodiments, the sequence of the first strand comprised by the first linker set is selected from any 2 to 20, 20 to 40, 40 to 60, 60 to 80, 80 to 96 of SEQ ID NO: 1 to SEQ ID NO: 96.

[0074] In certain embodiments, the second linker set comprises 2 to 30, 30 to 60, 60 to 90, 90 to 120, 120 to 150, 150 to 180, or 180 to 210 third strands.

[0075] In certain embodiments, the sequence of the third strand comprised by the second linker set is selected from any 2 to 20, 20 to 40, 40 to 60, 60 to 80, 80 to 96 of SEQ ID NO: 98 to SEQ ID NO: 193.

[0076] In certain embodiments, the third linker set comprises 2 to 30, 30 to 60, 60 to 90, 90 to 120, 120 to 150, 150 to 180, or 180 to 210 fourth strands.

[0077] In certain embodiments, the sequence of the fourth chain comprised by the third linker set is selected from any 2 to 20, 20 to 40, 40 to 60, 60 to 80, 80 to 96 of SEQ ID NO: 195 to SEQ ID NO: 290.

[0078] In certain embodiments, the fourth linker set comprises 2 to 30, 30 to 60, 60 to 90, 90 to 120, 120 to 150, 150 to 180, or 180 to 210 sixth strands.

[0079] In certain embodiments, the sequence of the sixth strand comprised by the fourth linker set is selected from any 2 to 20, 20 to 40, 40 to 60, 60 to 80, 80 to 96 of SEQ ID NO: 293 to SEQ ID NO: 388.

[0080] In certain embodiments, the linker sets each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 second strands, fifth strands or seventh strands.

[0081] In certain embodiments, the second strand has the sequence shown as SEQ ID NO:97.

[0082] In certain embodiments, the fifth strand has the sequence shown as SEQ ID NO:291.

[0083] In certain embodiments, the seventh strand has the sequence shown in SEQ ID NO:389.

[0084] Those skilled in the art will appreciate that the number of the first chain, the third chain, the fourth chain and the sixth chain in the joint group is not limited to the number specifically used in the embodiments of the present application. Using any 20 to 40, 40 to 60, 60 to 80, 80 to 96 of the 96 first chains, third chains, fourth chains or sixth chains provided in the present application can achieve the labeling of RNA in high-throughput single cell samples. Based on the 96 first chains, third chains, fourth chains or sixth chains provided in the present application, additional use of one or more first chains, third chains, fourth chains or sixth chains can also achieve the labeling of RNA in high-throughput single cell samples.

[0085] In certain embodiments, the number of the first chain, the third chain, the fourth chain, or the sixth chain in the linker set is determined based on the specific number of cells contained in the provided sample. For example, when the provided sample contains a large number of cells (e.g., 10 5 -10 6 ), then the 96 first chains, third chains, fourth chains or sixth chains provided in the present application can be used. For example, when the provided sample contains a relatively small number of cells (e.g., 50-100, 10 2 -10 3 , 10 3 -10 4 ), then any 20 to 40, 40 to 60, 60 to 80, 80 to 96 of the 96 first chains, third chains, fourth chains or sixth chains provided in the present application can be used.

[0086] Methods for labeling RNA from low-throughput single-cell samples

[0087] In a third aspect, the present application provides a method for labeling RNA of a single cell, the method comprising:

[0088] (1) providing a sample containing cells, and lysing the cells;

[0089] (2) fragmenting the RNA in the product obtained in step (1) to obtain multiple RNA fragments;

[0090] (3) adding a low-throughput adapter set to label the RNA fragments, wherein the low-throughput adapter set comprises one or more adapters.

[0091] In certain embodiments, in step (2), the 3' end of the RNA fragment is repaired to a hydroxyl group.

[0092] In certain embodiments, the low-throughput adapter set comprises 2 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100 adapters.

[0093] In certain embodiments, the sequences of the adapters included in the low-throughput adapter set are selected from any 2 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 42 of SEQ ID NO: 392 to SEQ ID NO: 433.

[0094] Those skilled in the art will appreciate that the number of joints included in the low-throughput joint group is not limited to the number specifically used in the embodiments of the present application. Using any 2 to 10 of the 42 joints provided in the present application, 10 to 20, 20 to 30, 30 to 40, 40 to 42 can achieve the labeling of RNA in low-throughput single cell samples. Based on the 42 joints provided in the present application, the additional use of 1 or more joints can also achieve the labeling of RNA in low-throughput single cell samples. In certain embodiments, the number of joints in the joint group is determined according to the specific cell number contained in the sample provided.

[0095] In a third aspect, the present application provides a method for obtaining RNA with a modification, wherein the method further comprises, after steps (1) to (3) of the aforementioned method:

[0096] (4) Providing an antibody that can specifically bind to the modified RNA, and contacting the antibody with the modified RNA.

[0097] In certain embodiments, after step (4), the remaining RNA is collected.

[0098] In certain embodiments, the modified RNA is selected from: 7 G modified RNA with m 5 C-modified RNA with m 6 A modified RNA, or any combination thereof.

[0099] In certain embodiments, prior to step (4), the labeled RNA obtained in step (3) is purified.

[0100] In certain embodiments, in step (2), the intracellular RNA is fragmented, the 5' end cap of the RNA is removed, and then the 3' end of the RNA fragment is repaired to a hydroxyl group.

[0101] In certain embodiments, the RNA 5' end cap is removed simultaneously with RNA shearing.

[0102] In certain embodiments, the RNA shearing method is selected from ion and high temperature heating methods or RNA enzymatic cleavage methods.

[0103] In certain embodiments, the method of 3' end repair is: contacting the broken RNA with a nucleic acid polymerase (eg, T4PNK enzyme);

[0104] In certain embodiments, the sample comprises 1, 2-5, 5-10, 10-20, 20-30, 30-40, 40-50 cells.

[0105] In certain embodiments, the sample is a sample obtained from a non-mammal (e.g., a zebrafish embryo), or a sample obtained from a mammal (e.g., a mouse, a human);

[0106] In certain embodiments, the RNA comprises mRNA, tRNA, rRNA, or any combination thereof.

[0107] In certain embodiments, the RNA comprises mRNA and / or tRNA.

[0108] Library for analyzing modification information carried by RNA and method for constructing library

[0109] In a fifth aspect, the present application provides a library for analyzing modification information carried by RNA, wherein the library is constructed by the following RNA samples:

[0110] (1) The modified RNA obtained by the method according to the second aspect; or

[0111] (2) The modified RNA obtained by the method according to the fourth aspect.

[0112] In certain embodiments, the modified RNA is selected from: 7 G modified RNA with m 5 C-modified RNA with m 6 A modified RNA, or any combination thereof.

[0113] In certain embodiments, the RNA comprises mRNA, tRNA, rRNA, or any combination thereof.

[0114] In certain embodiments, the RNA comprises mRNA and / or tRNA.

[0115] In a sixth aspect, the present application provides a method for constructing a library for analyzing modification information carried by RNA, wherein the method comprises: reverse transcribing an RNA sample into cDNA,

[0116] Wherein, the RNA sample is the modified RNA obtained in the aforementioned method; or is the modified RNA obtained in the aforementioned method.

[0117] In certain embodiments, the method comprises: reverse transcribing the RNA sample and performing PCR amplification; then performing T7-RNA polymerase amplification; then removing rRNA from the amplified product; then performing reverse transcription and PCR amplification; and optionally, purifying cDNA from the amplified product.

[0118] In certain embodiments, the modified RNA is selected from: 7 G modified RNA with m 5 C-modified RNA with m 6 A modified RNA, or any combination thereof.

[0119] In certain embodiments, the RNA comprises mRNA, tRNA, rRNA, or any combination thereof.

[0120] In certain embodiments, the RNA comprises mRNA and / or tRNA.

[0121] Transcriptome sequencing library and method for constructing library

[0122] In a seventh aspect, the present application provides a library for transcriptome sequencing, wherein the library is constructed by selecting any one of the following RNA samples:

[0123] (1) The labeled RNA obtained by the method according to the first aspect;

[0124] (2) the modified RNA obtained by the method of the second aspect, and the remaining RNA collected;

[0125] (3) The labeled RNA obtained by the method according to the third aspect;

[0126] (4) The modified RNA obtained by the method described in the fourth aspect, and the remaining RNA collected.

[0127] In certain embodiments, the modified RNA and the collected remaining RNA are each constructed into independent libraries.

[0128] In certain embodiments, the RNA comprises mRNA, tRNA, rRNA, or any combination thereof.

[0129] In certain embodiments, the RNA comprises mRNA and / or tRNA.

[0130] In an eighth aspect, the present application provides a method for constructing a library for transcriptome sequencing, wherein the method comprises: reverse transcribing an RNA sample into cDNA, wherein the RNA sample is selected from any one or more of the following:

[0131] (1) The labeled RNA obtained by the method according to the first aspect;

[0132] (2) the modified RNA obtained by the method of the second aspect, and the remaining RNA collected;

[0133] (3) The labeled RNA obtained by the method according to the third aspect;

[0134] (4) The modified RNA obtained by the method described in the fourth aspect, and the remaining RNA collected.

[0135] In certain embodiments, the method comprises: reverse transcribing the RNA sample and performing PCR amplification; then performing T7-RNA polymerase amplification; then removing rRNA from the amplified product; then performing reverse transcription and PCR amplification; and optionally, purifying cDNA from the amplified product.

[0136] In certain embodiments, the RNA comprises mRNA, tRNA, rRNA, or any combination thereof.

[0137] In certain embodiments, the RNA comprises mRNA and / or tRNA.

[0138] Analytical methods

[0139] In a ninth aspect, the present application provides a method for analyzing modification information carried by RNA, the method comprising: performing sequencing using the library as described above, and analyzing the data obtained from the sequencing.

[0140] In certain embodiments, the modified RNA is selected from: 7 G modified RNA with m 5 C-modified RNA with m 6 A modified RNA, or any combination thereof.

[0141] In certain embodiments, the RNA comprises mRNA, tRNA, rRNA, or any combination thereof.

[0142] In certain embodiments, the RNA comprises mRNA and / or tRNA.

[0143] In a tenth aspect, the present application provides a method for analyzing transcriptome information, the method comprising: performing sequencing using the library as described above, and analyzing the data obtained from the sequencing.

[0144] In the eleventh aspect, the present application provides a method for simultaneously analyzing transcriptome RNA and the modified RNA contained therein, the method comprising:

[0145] Establishing a library using the modified RNA obtained in the above method, and establishing a library using the remaining collected RNA, sequencing the two libraries, and analyzing the data obtained from the sequencing; or,

[0146] A library is established using the modified RNA obtained in the above-described method, and a library is established using the remaining collected RNA. The two libraries are sequenced, and the data obtained from the sequencing are analyzed.

[0147] In certain embodiments, the RNA comprises mRNA, tRNA, rRNA, or any combination thereof.

[0148] In certain embodiments, the RNA comprises mRNA and / or tRNA.

[0149] Reagent test kit

[0150] In a twelfth aspect, the present application provides a kit comprising the first linker group, the second linker group, the third linker group, the fourth linker group and / or the fifth linker group as defined in the first aspect or the second aspect.

[0151] In certain embodiments, the kit comprises a first linker set, a second linker set, a third linker set, a fourth linker set and a fifth linker set as defined in the first aspect or the second aspect.

[0152] In certain embodiments, the kit further comprises a low-throughput adaptor set as defined in the third aspect or the fourth aspect.

[0153] In certain embodiments, the kit further comprises: a reagent for adenylation (e.g., adenylase), a reagent for phosphorylation (e.g., T4 polynucleotide kinase), a DNA polymerase (e.g., T4 DNA ligase), a metal ion (e.g., zinc ion, magnesium ion), a reagent for cell fixation and permeabilization (e.g., formaldehyde, methanol, Digitonin, TritonX-100, Tween-20), a reagent for RNA shedding (e.g., ribonuclease (RNase I), micrococcal nuclease (MNase)), a reagent for RNA sequencing, a reagent for RNA 3' end repair, a reagent for sorting single cells, or any combination thereof.

[0154] In certain embodiments, the kit is used to implement the method of the first aspect or the second aspect and / or the method of the third aspect or the fourth aspect.

[0155] Definition of terms

[0156] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0157] As used herein, the terms "transcriptome" and "whole transcriptome" have the same meaning and can be used interchangeably. It refers to the collection of all transcription products in a cell under a certain physiological condition. Typically, the transcriptome includes messenger RNA, ribosomal RNA, transfer RNA and non-coding RNA. Transcriptome sequencing includes all mRNAs in a tissue or cell, regardless of whether the mRNA will be translated.

[0158] As used herein, the term "adenylation" refers to the use of adenylylase to decompose ATP into AMP and PPi using 5' phosphorylated single-stranded DNA as a substrate, and AMP is transferred to the 5' phosphate group of the single-stranded DNA to form adenylylated single-stranded DNA, thereby preparing an adenylylated linker.

[0159] As used herein, the term "phosphorylation" means that phosphorylation is a process in which a phosphate group is added to an intermediate metabolite or to a protein.

[0160] As used herein, the term "immunoprecipitation" refers to a method of purifying and enriching a target protein and its bound protein or nucleic acid substrate using an antibody-specific reaction. Generally speaking, immunoprecipitation first binds the antibody to the corresponding protein in the cell lysate or expression supernatant, then incubates with protein A / G (ProteinA / G) or secondary antibody-coupled beads (Agarose or Sepharose), and obtains a bead-protein A / G or secondary antibody-antibody-target protein complex by centrifugation. After washing, the precipitate can be used for purification and extraction of the precipitated product, including protein, DNA or RNA.

[0161] As used herein, the term "alcohol precipitation" refers to a method for removing impurities and retaining the effective components of interest. In this context, it refers to the use of ethanol to precipitate RNA, which utilizes the fact that nucleic acids are water-soluble compounds of polyanions and can combine with many monovalent and divalent ions to form salts, which are insoluble and non-denatured in organic solvents and form precipitates at relatively low temperatures, thereby removing other impurities.

[0162] As used herein, the term "neutralizing primer" refers to a primer that can be complementary to a joint group, i.e., all primers that can realize the function of neutralizing a joint group. In the method of the present application, in order to ensure the correct connection of the joint group, it is necessary to carry out saturated pairing consumption in advance with a neutralizing primer for the redundant joint group before mixing. It will be appreciated by those skilled in the art that the neutralizing primer of the present application is not limited to the neutralizing primer of the specific sequence actually used by the embodiment, as long as the primer that can realize the function of neutralizing a joint group belongs to the scope intended to be protected by the present application. Therefore, in certain embodiments, the first neutralizing primer is a primer that can be complementary to the first template sequence, the second template sequence or their partial sequences of the third chain. In certain embodiments, the first neutralizing primer is the second chain. In certain embodiments, the second neutralizing primer is a primer that can be complementary to the first template sequence, the second template sequence or their partial sequences of the fourth chain in the third joint group. In certain embodiments, the second neutralizing primer is the fifth chain. In certain embodiments, the third neutralizing primer is a primer that can be complementary to the first template sequence, the second template sequence or their partial sequences of the sixth chain in the fourth joint group. In certain embodiments, the third neutralizing primer is the seventh strand.

[0163] Advantageous Effects of the Invention

[0164] The inventors of the present application provide two uniquely designed methods for labeling RNA. The first method is suitable for single cells or low-throughput single cells (less than 50), i.e., low-throughput single-cell samples; the second method is suitable for a large number of cells (50 to 10 6The present inventors used immunoprecipitation to capture RNA with mRNA in the labeled RNA. 7 G-modified RNA, and sequence and analyze it to obtain mRNA of single-cell RNA in low-throughput cell samples and high-throughput cell samples. 7 G modification information.

[0165] In addition, the inventors of the present application unexpectedly found that in the labeled RNA, the capture of m 7 After the RNA modified with mG, the remaining RNA sample also has key information. 7 G-modified RNA samples were separately used to establish libraries and sequence them, and after integrating the sequencing data, it was found that it could reflect the transcriptome information. Therefore, the method of this application can reuse RNA samples with m 7 G modified RNA sample information, in order to use it to carry out 7 G-modified RNA analysis and transcriptome analysis, thus saving the time and economic cost of sequencing.

[0166] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0167] Figure 1 The flowchart and schematic diagram of the method for labeling RNA of cells suitable for high-throughput single-cell samples.

[0168] Figure 2 A diagram showing the complementary or connecting relationship of each chain in the first to fifth adapter groups in the method for labeling RNA suitable for high-throughput single-cell samples.

[0169] Figure 3 A diagram showing the complementary or connection sequences of each chain in the first to fifth adapter groups in the method for labeling RNA suitable for high-throughput single-cell samples.

[0170] Figure 4 The m of a single cell obtained by a method suitable for low-throughput single-cell samples 7 G modification group information, where Figure 4 A is m 7 Distribution characteristics of G modification peaks on mRNA, Figure 4 B is m 7The distribution ratio of G modification peaks in different regions of mRNA (5' untranslated region, coding region and 3' untranslated region), Figure 4 C is mRNA 7 Sequence preference of G modification peaks.

[0171] Figure 5 The single cell m obtained by the method of the present application 7 G modified sequencing (m 7 The number of reads of effective markers for G-IP).

[0172] Figure 6 For Input, Flow through and m 7 The sequencing results of the libraries established for the three groups of G-IP samples, among which, Figure 6 A is the result of sequencing data of three groups of samples without merging. Figure 6 B is to connect Flow through and m 7 Results after merging the G-IP data.

[0173] Figure 7 m is the m of a single cell obtained by a method suitable for high-throughput single-cell samples 7 G modification group information, where Figure 7 A is the distribution of the number of 9005 single-cell deduplication reads in the Input library before decapping. Figure 7 B is the statistical result of the number of deduplicated reads and effective genes of 9005 single cells in the Input library before decapping. Figure 7 C is the distribution of the number of 7992 single-cell deduplication reads in the Input library after decapping. Figure 7 D is the statistical result of the number of deduplicated reads and effective genes of 7992 single cells in the Input library after capping.

[0174] Sequence information

[0175] The information of the partial sequences involved in the present invention is provided in Table 1 below.

[0176] Table 1: Description of sequences

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] Note: "N" in the above table represents any one of the bases A, T, C, and G; the 5' ends of the first, third, fourth, and sixth chains are all phosphorylated; "*" represents thiolation modification. DETAILED DESCRIPTION

[0190] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0191] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Press): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)); and ANIMAL CELL CULTURE (Animal Cell Culture, 2nd edition). CELL CULTURE) (RI Freshney, ed. (1987)).

[0192] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0193] Example 1. Cell lysis, extraction or fixation

[0194] This application provides two technical solutions. The first technical solution is suitable for single cells or low-throughput single cells (less than 50), that is, low-throughput single-cell samples; the second technical solution is suitable for a large number of cells (50 to 10 6 The two sets of technical solutions are mainly different in the initial cell processing and labeling methods. Therefore, the experimental steps corresponding to the two technical solutions are provided in Examples 1-4.

[0195] Low-throughput single-cell samples :For single cells or low-throughput single cells (less than 50). After the cells are rinsed with DPBS, add cell lysis buffer (3.9μL RLT lysis buffer, 0.25μL RNAinhibitor, 0.3μL Glycogen, 0.05μLβ-mercaptoethanol, 20μL Absolute ethanol, 5.5μL Proteinase K) and lyse at room temperature for 10 minutes. Then, add 150μL 100% ethanol. Alcohol precipitation for 1 hour or overnight. Then centrifuge, remove the supernatant, and obtain RNA precipitation.

[0196] High-throughput single-cell samples :This scheme can mark 10 at the same time 6 cells. After the cells were rinsed twice with DPBS, 10 mL of 4% formaldehyde (1 mL of 4% formaldehyde was added for every 1 million cells) was added, and the cells were fixed on ice for 20 minutes, and mixed every 5 minutes. At the same time, 5% Digitonin was taken out in advance and preheated to melt at 90°C. 10 μL of 5% Digitonin and 30 μL of RNase Inhibitor were added to the fixed cells, and after gently mixing, they were placed on ice for 5 minutes, and mixed every 2 minutes. Finally, the cell permeability was evaluated by a cell counter.

[0197] Example 2. RNA shearing and 3' end repair

[0198] Low-throughput single-cell samples: Based on the above Example 1, 10 μl RNA shearing solution (9 μl 1×PFE buffer (Vazyme), 0.5 μl Turbo DNase I) was added, placed in a PCR instrument, reacted at 42°C for 10 min, 85°C for 10 min, 75°C for 10 min, and finally kept constant at 4°C. The RNA was sheared into fragments of about 100 nt. After the reaction, 40.5 μl 5'mRNA cap removal solution (5 μl 10×Thermopol Buffer NEB (B9004S), 2 μl RNA5'Pyrophosphohydrolase (RppH), 33.5 μl NF·H2O) was directly added on the basis of the above, reacted at 37°C for 60 min, and then reacted at 65°C for 5 min. After the above reaction was completed, 180 μl 100% ethanol was added for alcohol precipitation.

[0199] High-throughput single-cell samples :Prepare solution A, that is, add 500μL 10mM Tris-HCL (pH 8.0) and 20μL 5% Tritonx-100 to 500μL DPBS. Based on the above Example 1, the permeabilized cells are divided into two tubes, each with 5mL, and centrifuged at 500g for 5 minutes in a constant temperature high-speed centrifuge precooled at 4℃. Carefully remove the supernatant without touching the cell pellet, add 500μL solution A to each tube, mix well and transfer to a 1.5mL centrifuge tube. Centrifuge again at the same speed for 3 minutes, remove the supernatant, add 500μL solution A, mix well and centrifuge to remove the supernatant. At this time, add an equal volume of solution A (100μL), and then add 20μL 10×Fragmentation reagent, and react in a 37℃ metal bath for 90 minutes. After the reaction, add 20μL stop solution and 20μL solution A, mix thoroughly, centrifuge at 500g for 3 minutes in a 4℃ precooled thermostatic centrifuge, remove the supernatant, add 500μL solution A, mix well, centrifuge again, and remove the supernatant. After the reaction, directly add 50μL 5'mRNA cap removal solution (5μL 10×ThermopolBuffer NEB(B9004S),3μL RNA 5'Pyrophosphohydrolase(RppH),42μL NF·H2O) on the basis of the above, and react at 37℃ for 60min. After the above reaction, centrifuge at 500g for 3 minutes in a 4℃ precooled thermostatic centrifuge, remove the supernatant, add 500μL solution A, mix well, centrifuge again, and remove the supernatant.

[0200] Example 3.3' End repair

[0201] Low-throughput single-cell samples Based on the above Example 2, high-speed centrifugation was performed for 40 min to obtain RNA fragment precipitation. 11.9 μL NF·H2O, 1.6 μL T4 PNK reaction buffer (NEB, M0201L), 2 μL T4 PNK enzyme, and 0.5 μL RNase Inhibitor were added to the fragmented RNA sample. After being fully mixed, the mixture was placed in a PCR instrument at 37° C. for 1 hour to repair the 3′ end of the RNA fragment to a hydroxyl group for connection to the adapter sequence.

[0202] High-throughput single-cell samples :Based on the above Example 2, 450 μL of NF·H2O, 50 μL of T4 PNK buffer, 20 μL of 5% TritonX-100, and 20 μL of T4PNK enzyme were added to each tube, and reacted in a 37°C metal bath for 90 minutes. After the above reaction, centrifuge at 500g for 3 minutes in a constant temperature centrifuge precooled at 4°C, remove the supernatant, add 500 μL of solution A, mix well, centrifuge again, and remove the supernatant.

[0203] Example 4. Single cell labeling

[0204] Low-throughput single-cell samples Based on the above Example 3, 18 μL T4 RNA ligase solution (200 μL 10×T4 RNA Ligase Reaction Buffer, 600 μL 50% PEG8000*, 50 μL T4 RNA Ligase 2truncated KQ, 200 μL NF·H2O) and 2 μL Adaptor (20 μM) (a total of 42 sequences, the specific sequences are shown in SEQ ID NO:392 to SEQ ID NO:433) were added to the reaction, placed in a PCR instrument, and reacted at 25°C for 1 h and 16°C overnight.

[0205] High-throughput single-cell samplesBased on the above Example 3, the cells after 3' end repair were synthesized into one tube, 100 μL T4 DNA ligase buffer was added, and the mixture was thoroughly mixed and divided into 10 tubes, each with 100 μL. Then, 10 μL of the first linker group R1 consisting of the first chain shown in SEQ ID NO: 1 to SEQ ID NO: 45 and the second chain shown in SEQ ID NO: 97 was added (the first linker group was pre-adenylated by 5'DNA Adenylation Kit (NEB#E2610L)), and the reaction was carried out in a metal bath at 37°C overnight. After the reaction, add 200 μL of solution A respectively, mix thoroughly, centrifuge at 600g for 3 minutes, carefully remove the supernatant, add 500 μL of solution A to the first tube, mix thoroughly and add to the second tube, repeat the process until the 10th tube, mix thoroughly, divide into 10 tubes, add 3 μL of the second linker group R2 composed of the third chain shown in SEQ ID NO: 98 to SEQ ID NO: 193 to each tube, and react in a 37°C metal bath for 30-60 minutes, preferably 45 minutes. After the reaction, 3.2 μL of the second chain was added to neutralize the excess third chain. After reacting in a 37°C metal bath for 15 minutes, 10 tubes were collected into 1 tube in the previous manner, and 90 μL of T4 DNA ligase was added. After sufficient mixing, the tubes were divided into 10 tubes, each with 100 μL. 3.5 μL of the third linker group R3 consisting of the fourth chain shown in SEQ ID NO: 195 to SEQ ID NO: 290 and the fifth chain shown in SEQ ID NO: 291 were added respectively. The reaction was carried out in a 37°C metal bath for 45 minutes. 3.8 μL of the fifth chain was added respectively to neutralize the excess fourth chain. After mixing, the mixture was reacted in a 37°C metal bath for 15 minutes and the tubes were also collected into 1 tube. Then add 90 μL T4 DNA ligase, mix well and dispense into 10 tubes, 100 μL in each tube, then add 4 μL of the fourth linker group R4 composed of the sixth chain shown in SEQ ID NO:293 to SEQ ID NO:388 and the seventh chain shown in SEQ ID NO:389, respectively, react in a 37°C metal bath for 45 minutes, then add 4.3 μL of the seventh chain to neutralize the excess sixth chain, react in a 37°C metal bath for 15 minutes, and collect into 1 tube. Divide into two tubes, 500 μL in each tube, centrifuge at 500g for 3 minutes in a centrifuge, and remove the supernatant. Add appropriate amounts of dNTP mix (10mM), Bst DNA polymerase, 10XIsothermal Amplification Buffer and RNase Inhibitor, the total volume is 500 μL, dispense into 8 strip tubes, 25 μL in each tube, and add 2.5 μL of different eighth chains (i.e., Figure 2 The P5 connector in the tube was purchased from NEB, the index of E7500L NEBNext Multiplex Oligos for Illumina.E7500L, and placed in a metal bath at 37°C for 30 minutes. After the reaction, one tube was synthesized and then divided into two tubes. Centrifuged at 500g for 3 minutes in a centrifuge, the supernatant was removed, 500μL solutionA was added to each tube, filtered with a 40μm filter membrane, and collected in a flow tube for single cell sorting. Every 10,000 single cells were sorted into a 1.5mL centrifuge tube with 50μL lysis solution.

[0206] Example 5.m 7 G-modified RNA capture

[0207] (1) Purification of labeled RNA

[0208] Based on the above Example 4, the labeled RNA sample was purified by RNA clean beads to remove residual enzymes, salt ions and excess barcode sequences in the reaction system. Finally, 180 μL NF·H2O was used to elute the sample (if multiple labeled single cells were combined, the total volume of eluted NF·H2O was 180 μL).

[0209] (2) Prepare m 7 G-protein A / G coupled magnetic beads

[0210] Take out 10μL Protein A / G beads into a 1.5mL centrifuge tube, add 200μL 1×IP buffer (10mM Tris-HCl (pH 7.4), 150mM NaCl, 0.1% NP-40), mix well and place on a magnetic rack, discard the supernatant, and repeat washing 3 times. Then add 100μL 1×IP buffer and 5μg m 7 G antibody (MBL, cat: RN017M), incubate with rotation at 4°C overnight. 7 After incubation with Protein A / G beads, place them on a magnetic rack, wait for the solution to clarify, discard the supernatant, rinse three times with 1×IP buffer, and finally add 20μL 10×IP buffer and mix well.

[0211] (3) Immunoprecipitation

[0212] 1 / 10 of the RNA obtained in Example 5 (1) was retained as input (for subsequent single-cell transcriptome library construction, if the sample is too small, it can be omitted), and the m obtained in Example 5 (2) was 7The G-protein A / G coupled magnetic beads mixture was added to 9 / 10 of the RNA, followed by 3 μL RNase Inhibitor and 2 μL Cocktail, and the reaction was rotated at 4°C for 6 hours.

[0213] (4) Obtain Flow through samples

[0214] After the reaction in Example 5 (3) is completed, the sample is placed on a magnetic rack and the supernatant is collected as a flow-through sample (for subsequent single-cell transcriptome library construction).

[0215] (5) Washing

[0216] Method 1. After the reaction is completed as in Example 5 (4), the cells were washed once with 1×IP buffer, twice with High salt buffer (10 mM Tris-HCl (pH 7.4), 500 mM NaCl, 0.1% NP-40), and once with 1×PK buffer (100 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM EDTA). Each wash was performed with rotation at 4°C for 2 minutes.

[0217] Method 2: To enhance m 7 G antibody and target m 7 The binding strength of G-modified RNA was determined and the m 7 G site information. Example 5 (4) After the reaction was completed, the magnetic beads were resuspended in 1×IP buffer and heated at 254 nm at 0.15 J / cm 2 After the reaction, the beads were collected into a 1.5 mL EP tube and placed on a magnetic stand. After the solution was clarified, the supernatant was discarded and the beads were rinsed once with 1×IP buffer, twice with High salt buffer (10 mM Tris-HCl (pH 7.4), 500 mM NaCl, 0.1% NP-40), and then once with 1×PK buffer (100 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM EDTA). Each wash was rotated at 4°C for 2 minutes.

[0218] In a specific implementation scheme, this example is an experiment performed by the scheme of method one.

[0219] (6) Elution

[0220] Finally, resuspend the beads in 280 μL 1×PK buffer, add 20 μL Proteinase K, and react in a thermostat at 55°C, 1500 rpm for 1 h.

[0221] After the reaction was completed, the reaction mixture was placed back on the magnetic rack for 2 minutes and the supernatant was collected. An equal volume of phenol chloroform (pH < 5.0) was added to the supernatant and the subsequent alcohol precipitation yielded m 7 G-IP samples.

[0222] Example 6. Single Cell m 7 G modification group and transcriptome library construction

[0223] (1) Reverse transcription

[0224] In Example 5, Input, Flow through and m 7 In the G-IP group, reverse transcription reagent and reverse transcriptase SMARTScribe were added. TM Reverse Transcriptase (Clontech, Cat: #639538), add reverse transcription primer Pico RT and strand displacement primer N4TSO (sequence information is the same as patent: A method for labeling and sequencing the entire transcriptome of intracellular RNA (application number: 202111516580.6)), vortex and oscillate to mix thoroughly, place in a PCR instrument, 25°C for 5 min, 42°C for 90 min, 85°C for 10 min, and store at 4°C.

[0225] (2)1 st PCR amplification

[0226] The cDNA obtained above was purified with DNA clean beads and dissolved in 32 μL NF·H2O. PicoRT and T7-pre-universal primers (sequence information is the same as patent: A method for labeling and sequencing the entire transcriptome of intracellular RNA (application number: 202111516580.6)) were used to amplify the entire transcriptome. For single-cell samples, 18 cycles of amplification of the entire transcriptome library were required.

[0227] (3) T7 amplification

[0228] According to the instructions of the T7 High Efficiency Transcription Kit (TransGen Biotech, Cat: #JT101-01), the T7 reverse transcription system was prepared, vortexed and mixed, and then reacted in a PCR instrument at 37°C overnight. After the reaction was completed, 2μL EDTA was added to terminate the reaction, and the RNA was purified with RNAclean beads and dissolved in 80μL enzyme-free water to measure the concentration of RNA. After removing the template DNA in the reaction system with Turbo DNase, it was purified again with RNA clean beads and finally dissolved with 40μL NF·H2O.

[0229] (4) Removal of rRNA

[0230] The RNA obtained in Example 6 (3) above was added to 11 μL 5× hybridization buffer (1M NaCl, 500mM Tris-HCl, pH 7.4), 2 μL rRNA probe (depending on the species), and 2 μL RNase Inhibitor. The reaction was placed on a PCR instrument: 95°C for 2 min, 95°C cooled by 1°C every 10s until 22°C, 22°C for 5 min, and stored at 4°C. Subsequently, RNase H and TURBO DNase were used to remove the hybrid chains and excess probes of rRNA. Finally, RNA clean beads were used for purification, and finally, 30 μL NF·H2O was used for dissolution.

[0231] (5) Reverse transcription and 2 nd PCR amplification

[0232] Pico-RT primer was added to the RNA system obtained in Example 6 (4) above, and reverse transcription was performed according to the instructions of HiFiScript gDNARemoval cDNA Synthesis Kit (CWBIO).

[0233] After reverse transcription, RNase A was added and digested at 37°C for 5 min. After purification with DNA clean beads, the DNA was dissolved in 32 μL NF·H2O.

[0234] Finally, use Index primers and universal primers (sequence information is the same as the patent: A method for labeling and sequencing the entire transcriptome of intracellular RNA (application number: 202111516580.6)) to perform PCR amplification according to the KAPA HiFi HotStart ReadyMix (KAPABiosystem, KK2602) kit, which requires 6-8 cycles.

[0235] After PCR, the DNA was purified again with DNA clean beads and dissolved in 20 μL NF·H2O to obtain a library for sequencing.

[0236] Example 7. Sequencing and data analysis

[0237] Laboratory-generated RNA mRNAs from single cells at different developmental stages 7 The G library was sequenced on the Nova-PE150 platform for double-end sequencing. The raw data obtained from the sequencing machine were processed and analyzed using a variety of bioinformatics software.

[0238] (1) Sequencing data removal of adapters and PCR duplicates

[0239] The software cutadapt was used to remove the sequencing adapter sequences in the original sequencing data, and the written perl script was used to filter the PCR amplified repetitive sequences according to the base information of the obtained data.

[0240] (2) Sequencing data sorting and base filtering

[0241] After data sorting based on the barcode sequence at the R2 end to identify the sample source, the software Trimmomatic was used to filter and remove reads with base quality less than 20 and length less than 35 nt. The software FastQC evaluated the data quality of each quality control step.

[0242] (3) Sequencing data alignment and low match filtering

[0243] The hisat2 software was then used to align the quality-controlled sequencing data with the reference genome sequence, with the zebrafish zv9 version as the reference genome (parameter: -xU), and the samtools software was used to filter out reads with low alignment quality.

[0244] Note: The above processing (1)-(3) is applicable to Input, Flow through and m 7 G-IP Group.

[0245] (4) Modification site identification and mapping

[0246] Related 7 The G-IP group data and the Input group data were enriched and analyzed for modification sites using the MACS2 software tool. 7 The region that was significantly enriched in the G-IP group compared with the Input group was defined as m 7 G modification site; at the same time, associated m 7The G-IP group data and the Flow through group data were enriched and analyzed for modification sites using the MACS2 software tool. 7 G modification site. 7 G modification sites take the intersection as a more reliable m 7 G modification sites were used for downstream analysis.

[0247] The single-cell RNA mRNAs of zebrafish embryos at different stages were detected. 7 The number, distribution characteristics, and methylation levels of G sites were analyzed to analyze the RNA m 7 The distribution pattern, distribution motif, and methylation level changes of G sites were analyzed, and the RNA m 7 G map.

[0248] Experimental results:

[0249] For low-throughput single-cell samples We used a single zebrafish embryo blastomere (2-cell stage sample) and mixed blastomeres of different stages that had undergone one round of labeling (egg, 1-cell, 2-cell, and 4-cell stage samples) to perform single-cell m 7 G and transcriptome sequencing. Experimental data show that ( Figure 4 ), this method can successfully capture the m of single cells 7 G modifies group information.

[0250] To verify the single cell m 7 In order to test the specific labeling efficiency of single embryonic cells with the new technology of simultaneous detection of G and transcriptome, we performed single-cell labeling on samples from four periods: oocyte, 1-cell, 2-cell, and 4-cell (the number of cells used in each period is 1, 1, 2, and 4, a total of 8 cells, that is, there are 8 Barcode sequences represented). After labeling, the samples from each period were mixed together and then single-cell labeling was performed. 7 G and transcriptome sequencing. Through analysis, it was found that in the single-cell transcriptome sequencing data (Input: RNA-seq), the number of effectively labeled reads accounted for more than 85% of the total number, and the labeled reads were distributed in embryonic cells at all stages, indicating that this labeling technology is feasible for labeling in the early development of zebrafish embryos; 7 G modified sequencing (m 7 The results of G-IP) data showed that the number of effectively labeled reads accounted for more than 70% of the total ( Figure 5 ). The above results show that the transcriptome of a single cell and m 7The sequencing method for simultaneous detection of G modification groups can effectively and specifically mark each cell, and this result provides a basis for subsequent mapping of single cell m 7 G modification map laid the foundation.

[0251] Input, Flow through and m 7 G-IP library construction was performed in three groups of samples. First, the three groups of data were analyzed for composition. It was found that the samples were 7 G-IP was clustered into three categories, indicating that modification data provided epigenetic information different from single-cell transcriptome sequencing. 7 After the G-IP data were merged and reanalyzed, it was found that the merged data and the Input were clustered together ( Figure 6 ), indicating Flow through and m 7 G-IP can replace Input, which can save RNA used for Input library construction in subsequent studies, and facilitates the construction of libraries for precious samples and small amounts of samples.

[0252] For high-throughput single-cell samples : We marked 10 6 A preliminary experiment was conducted with HeLa cells, and five rounds of specific Barcode labeling were performed, with 10 specific Barcodes labeled in each group. Data analysis showed that the number of effectively labeled data before decapping was about 9,000, while the number of effectively labeled data after decapping was about 8,000 ( Figure 7 ). In general, the cell number throughput of the labeled cells is similar to that obtained by 10×Genomics transcriptome sequencing, which can be used for downstream preliminary analysis. In addition, the number of effectively labeled cells obtained at this stage can be increased by further improving the effective labeling rate of RNA and the first barcode, thus enabling population single-cell sequencing with a larger cell throughput.

[0253] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A method for labeling RNA of a single cell, the method include: (I) providing a sample containing cells, and fixing and / or permeabilizing the cells; (II) fragmenting the intracellular RNA to obtain multiple RNA fragments; (III) labeling the RNA fragments, the specific steps are as follows: (a) providing a first linker set, wherein the first linker set comprises at least one first chain and a second chain, The first strand comprises a linker sequence and a template sequence, wherein the linker sequence is a random arrangement of 6 to 12 (e.g., 6, 7, 8, 9, 10, 11, 12) bases, and each first strand contains a different linker sequence and the same template sequence; the second strand comprises a first template sequence and a second template sequence, and the first template sequence of the second strand is complementary to the template sequence of the first strand, Optionally, adenylase is contacted with the first strand, and the first linker group is contacted with the RNA fragment under conditions that allow nucleic acid hybridization or annealing, so that the first strand of the first linker group is connected to the 3' end of the RNA fragment; (b) providing a second linker set, wherein the second linker set comprises at least one third chain, The third strand comprises a first template sequence, a linker sequence and a second template sequence, the linker sequence is a random arrangement of 6 to 12 (e.g., 6, 7, 8, 9, 10, 11, 12) bases, and each third strand contains a different linker sequence and the same first template sequence and second template sequence; the first template sequence of the third strand is complementary to the second template sequence of the second strand in the first linker group, contacting the second linker set with the product of step (a) under conditions that allow nucleic acid hybridization or annealing, so that the second linker set is ligated to the 3' end of the first strand of the first linker set; Optionally, contacting a first neutralizing primer (e.g., the second strand) with the product obtained after the above annealing under conditions that allow nucleic acid hybridization or annealing, wherein the first neutralizing primer (e.g., the second strand) is complementary to the first template sequence, the second template sequence, or a partial sequence thereof of the third strand; (c) providing a third linker set, wherein the third linker set comprises at least one fourth chain and a fifth chain, The fourth strand comprises a first template sequence, a linker sequence and a second template sequence, the linker sequence is a random arrangement of 6 to 12 (e.g., 6, 7, 8, 9, 10, 11, 12) bases, and each fourth strand contains a different linker sequence and the same first template sequence and second template sequence, The fifth strand comprises a first template sequence and a second template sequence, wherein the first template sequence of the fifth strand is complementary to the second template sequence of the third strand, and the second template sequence of the fifth strand is complementary to the first template sequence of the fourth strand, contacting the third linker set with the product of step (b) under conditions that allow nucleic acid hybridization or annealing, so that the fourth strand of the third linker set is ligated to the 3' end of the second linker set; Optionally, under conditions that allow nucleic acid hybridization or annealing, contacting a second neutralizing primer (e.g., the fifth strand) with the product obtained after the above annealing, wherein the second neutralizing primer (e.g., the fifth strand) is complementary to the first template sequence, the second template sequence, or a partial sequence thereof of the fourth strand in the third adapter set; (d) providing a fourth linker set, wherein the fourth linker set comprises at least one sixth chain and a seventh chain, The sixth strand comprises a first template sequence, a linker sequence and a second template sequence, the linker sequence is a random arrangement of 14 to 20 (e.g., 14, 15, 16, 17, 18, 19, 20) bases, and each sixth strand contains a different linker sequence and the same first template sequence and second template sequence, The seventh strand comprises a first template sequence and a second template sequence, wherein the first template sequence of the seventh strand is complementary to the second template sequence of the fourth strand, and the second template sequence of the seventh strand is complementary to the first template sequence of the sixth strand, contacting the fourth linker set with the product of step (c) under conditions that allow nucleic acid hybridization or annealing, so that the sixth strand of the fourth linker set is linked to the 3' end of the fourth strand of the third linker set; Optionally, under conditions allowing nucleic acid hybridization or annealing, contacting a third neutralizing primer (e.g., the seventh strand) with the product obtained after the above annealing, wherein the third neutralizing primer (e.g., the seventh strand) is complementary to the first template sequence, the second template sequence, or a partial sequence thereof of the sixth strand in the fourth adapter set; (e) providing a fifth linker set, wherein the fifth linker set comprises at least one eighth chain, The eighth strand comprises a first template sequence, a linker sequence and a second template sequence, the linker sequence is a random arrangement of 6 to 12 (e.g., 6, 7, 8, 9, 10, 11, 12) bases, and each eighth strand contains a different linker sequence and the same first template sequence and second template sequence; the first template sequence of the eighth strand is complementary to the second template sequence of the sixth strand, contacting the fifth linker set with the product of step (d) under conditions that allow nucleic acid hybridization or annealing, so that the fifth linker set is linked to the 3' end of the sixth strand of the fourth linker set; (f) contacting a DNA polymerase with the product obtained in step (g) under conditions that allow nucleic acid ligation; (IV) sorting single cells to obtain RNA-labeled single cells; optionally, lysing the single cells to obtain labeled RNA.

2. A method for obtaining RNA with modification, in, The method further comprises, after steps (I) to (IV) of the method of claim 1: (V) providing an antibody that can specifically bind to the modified RNA, and contacting the antibody with the modified RNA; For example, providing a method capable of specifically binding to m 7 G modified RNA antibody, so that the antibody with m 7 G modified RNA contacts; Preferably, the modified RNA is selected from: 7 G modified RNA with m 5 C-modified RNA with m 6 A modified RNA, or any combination thereof; Preferably, after step (V), the remaining RNA is collected; Preferably, prior to step (V), the labeled RNA obtained in step (IV) is purified.

3. The method according to claim 1 or 2, in, The method has one or more characteristics selected from the following: (1) In step (II), the intracellular RNA is fragmented, the 5' end cap of the RNA is removed, and then the 3' end of the RNA fragment is repaired to a hydroxyl group; Preferably, the RNA shearing method is selected from ion and high temperature heating method or RNA enzymatic cleavage method; Preferably, the method for 3' end repair is: contacting the interrupted RNA with a nucleic acid polymerase (eg, T4 PNK enzyme); (2) in step (IV), single cells are sorted by flow cytometry; (3) The sample contains a plurality of cells, for example, 50 to 10 2 cells, 10 2 to 10 3 cells, 10 3 to 10 4 cells, 10 5 to 10 6 cells; (4) The sample is a sample obtained from a non-mammal (e.g., a zebrafish embryo) or a sample obtained from a mammal (e.g., a mouse, a human); (5) in steps (b) to (d), providing a DNA polymerase (e.g., a DNA ligase, e.g., T4 DNA ligase); (6) The RNA comprises mRNA, tRNA, rRNA, or any combination thereof; preferably, the RNA comprises mRNA and / or tRNA.

4. The method according to any one of claims 1 to 3, having one or more features selected from the following: (1) In step (a), the linker sequence is a random arrangement of 9 bases; (2) In steps (b) and (c), the connection sequence is a random arrangement of 8 bases; (3) In step (d), the linker sequence is a random arrangement of 16 bases; (4) In step (e), the linker sequence is a random arrangement of 8 bases; (5) the length of the template sequence of the first strand is 16-22 bases (e.g., 19 bases); (6) the length of the first template sequence of the second strand is 16-22 bases (e.g., 19 bases); (7) the length of the second template sequence of the second strand is 13-18 bases (e.g., 15 bases); (8) The length of the first template sequence of the third strand is 13-18 bases (e.g., 15 bases); (9) the length of the second template sequence of the third strand is 13-18 bases (e.g., 15 bases); (10) the length of the first template sequence of the fourth strand is 13-18 bases (e.g., 15 bases); (11) the length of the second template sequence of the fourth strand is 13-18 bases (e.g., 15 bases); (12) the length of the first template sequence of the fifth strand is 13-18 bases (e.g., 15 bases); (13) the length of the second template sequence of the fifth strand is 13-18 bases (e.g., 15 bases); (14) the length of the first template sequence of the sixth strand is 13-18 bases (e.g., 15 bases); (15) the length of the second template sequence of the seventh strand is 18-22 bases (e.g., 20 bases); (16) The length of the first template sequence of the eighth strand is 20-24 bases (e.g., 22 bases); (17) The length of the second template sequence of the eighth chain is 30-36 bases (e.g., 33 bases).

5. The method according to any one of claims 1 to 4, in, The method has one or more characteristics selected from the following: (1) the first linker set comprises 2 to 30, 30 to 60, 60 to 90, 90 to 120, 120 to 150, 150 to 180, or 180 to 210 first strands; (2) the sequence of the first chain contained in the first linker group is selected from any 2 to 20, 20 to 40, 40 to 60, 60 to 80, 80 to 96 of SEQ ID NO: 1 to SEQ ID NO: 96; (3) the second linker set comprises 2 to 30, 30 to 60, 60 to 90, 90 to 120, 120 to 150, 150 to 180, or 180 to 210 third strands; (4) the sequence of the third strand contained in the second linker group is selected from any 2 to 20, 20 to 40, 40 to 60, 60 to 80, 80 to 96 of SEQ ID NO: 98 to SEQ ID NO: 193; (5) the third joint group comprises 2 to 30, 30 to 60, 60 to 90, 90 to 120, 120 to 150, 150 to 180, or 180 to 210 fourth strands; (6) the sequence of the fourth chain contained in the third linker group is selected from any 2 to 20, 20 to 40, 40 to 60, 60 to 80, 80 to 96 of SEQ ID NO: 195 to SEQ ID NO: 290; (7) the fourth joint group comprises 2 to 30, 30 to 60, 60 to 90, 90 to 120, 120 to 150, 150 to 180, or 180 to 210 sixth chains; (8) the sequence of the sixth strand contained in the fourth linker group is selected from any 2 to 20, 20 to 40, 40 to 60, 60 to 80, 80 to 96 of SEQ ID NO: 293 to SEQ ID NO: 388; (9) the linker groups each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 second strands, fifth strands or seventh strands; (10) the second strand has the sequence shown in SEQ ID NO:97; (11) the fifth strand has the sequence shown in SEQ ID NO: 291; (12) The seventh chain has the sequence shown in SEQ ID NO:

389.

6. A method for labeling RNA of a single cell, the method include: (1) providing a sample containing cells, and lysing the cells; (2) fragmenting the RNA in the product obtained in step (1) to obtain multiple RNA fragments; (3) adding a low-throughput adapter set to label the RNA fragments, wherein the low-throughput adapter set comprises one or more adapters; Preferably, in step (2), the 3' end of the RNA fragment is repaired to a hydroxyl group; Preferably, the low-throughput adapter set comprises 2 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100 adapters; Preferably, the sequences of the adapters contained in the low-throughput adapter set are selected from any 2 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 42 of SEQ ID NO: 392 to SEQ ID NO:

433.

7. A method for obtaining RNA with modification, in, The method further comprises, after steps (1) to (3) of the method of claim 6: (4) providing an antibody that can specifically bind to the modified RNA, and contacting the antibody with the modified RNA; For example, providing a method capable of specifically binding to m 7 G modified RNA antibody, so that the antibody with m 7 G modified RNA contacts; Preferably, after step (4), the remaining RNA is collected; Preferably, the modified RNA is selected from: 7 G modified RNA with m 5 C-modified RNA with m 6 A modified RNA, or any combination thereof; Preferably, before step (4), the labeled RNA obtained in step (3) is purified.

8. The method according to claim 6 or 7, in, The method has one or more characteristics selected from the following: (1) In step (2), the intracellular RNA is disrupted, the 5' end cap of the RNA is removed, and then the 3' end of the RNA fragment is repaired to a hydroxyl group; Preferably, the RNA shearing method is selected from ion and high temperature heating method or RNA enzymatic cleavage method; Preferably, the method for 3' end repair is: contacting the interrupted RNA with a nucleic acid polymerase (eg, T4 PNK enzyme); (2) the sample contains 1, 2-5, 5-10, 10-20, 20-30, 30-40, 40-50 cells; (3) The sample is a sample obtained from a non-mammal (e.g., a zebrafish embryo) or a sample obtained from a mammal (e.g., a mouse, a human); (4) The RNA comprises mRNA, tRNA, rRNA, or any combination thereof; preferably, the RNA comprises mRNA and / or tRNA.

9. A library for analyzing modification information carried by RNA, wherein the library is constructed using the following RNA samples: (1) The modified RNA obtained by the method of any one of claims 2 to 5; or (2) The modified RNA obtained by the method of claim 7 or 8; Preferably, the modified RNA is selected from: 7 G modified RNA with m 5 C-modified RNA with m 6 A modified RNA, or any combination thereof; Preferably, the RNA comprises mRNA, tRNA, rRNA, or any combination thereof; Preferably, the RNA comprises mRNA and / or tRNA.

10. Method for constructing a library for analyzing modification information carried by RNA, in, The method comprises: reverse transcribing the RNA sample into cDNA, Wherein, the RNA sample is the modified RNA obtained by the method of any one of claims 2 to 5; or is the modified RNA obtained by the method of claim 7 or 8; Preferably, the method comprises: reverse transcribing the RNA sample and performing PCR amplification; then performing T7-RNA polymerase amplification; then removing rRNA from the amplified product; then performing reverse transcription and PCR amplification; optionally, purifying cDNA from the amplified product; Preferably, the modified RNA is selected from: 7 G modified RNA with m 5 C-modified RNA with m 6 A modified RNA, or any combination thereof; Preferably, the RNA comprises mRNA, tRNA, rRNA, or any combination thereof; Preferably, the RNA comprises mRNA and / or tRNA.

11. A library for transcriptome sequencing, wherein the library is constructed by selecting any one of the following RNA samples: (1) The labeled RNA obtained by the method of claim 1; (2) The modified RNA obtained by the method of any one of claims 2 to 5, and the remaining RNA collected; (3) The labeled RNA obtained by the method of claim 6; (4) The modified RNA obtained by the method of claim 7 or 8, and the remaining RNA collected; Preferably, the modified RNA and the collected remaining RNA are each used to construct an independent library; Preferably, the RNA comprises mRNA, tRNA, rRNA, or any combination thereof; Preferably, the RNA comprises mRNA and / or tRNA.

12. Method for constructing a library for transcriptome sequencing, in, The method comprises: reverse transcribing an RNA sample into cDNA, wherein the RNA sample is selected from any one or more of the following: (1) The labeled RNA obtained by the method of claim 1; (2) The modified RNA obtained by the method of any one of claims 2 to 5, and the remaining RNA collected; (3) The labeled RNA obtained by the method of claim 6; (4) The modified RNA obtained by the method of claim 7 or 8, and the remaining RNA collected; Preferably, the method comprises: reverse transcribing the RNA sample and performing PCR amplification; then performing T7-RNA polymerase amplification; then removing rRNA from the amplified product; then performing reverse transcription and PCR amplification; optionally, purifying cDNA from the amplified product; Preferably, the RNA comprises mRNA, tRNA, rRNA, or any combination thereof; Preferably, the RNA comprises mRNA and / or tRNA.

13. A method for analyzing modification information carried by RNA, the method include: Performing sequencing using the library described in claim 9, and analyzing the data obtained from the sequencing; Preferably, the modified RNA is selected from: 7 G modified RNA with m 5 C-modified RNA with m 6 A modified RNA, or any combination thereof; Preferably, the RNA comprises mRNA, tRNA, rRNA, or any combination thereof; Preferably, the RNA comprises mRNA and / or tRNA.

14. A method for analyzing transcriptome information, the method include: The library according to claim 11 is used for sequencing, and the data obtained from the sequencing are analyzed.

15. A method for simultaneously analyzing transcriptome RNA and modified RNA contained therein, the method comprising: include: Establishing a library using the modified RNA obtained by the method of any one of claims 2 to 5, and establishing a library using the remaining collected RNA, sequencing the two libraries, and analyzing the data obtained by sequencing; or, Establishing a library using the modified RNA obtained by the method of claim 7 or 8, and establishing a library using the remaining collected RNA, sequencing the two libraries, and analyzing the data obtained by sequencing; Preferably, the RNA comprises mRNA, tRNA, rRNA, or any combination thereof; Preferably, the RNA comprises mRNA and / or tRNA.

16. A kit comprising the first linker set, the second linker set, the third linker set, the fourth linker set and / or the fifth linker set as defined in any one of claims 1 to 4; Preferably, the kit further comprises a low-throughput adapter set as defined in any one of claims 5 to 8; Preferably, the kit is used to implement the method according to any one of claims 1 to 4 and / or the method according to any one of claims 5 to 8.

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