WO2021042883A1 - Method and kit for detecting rna n6-methyladenine modification at single-base resolution in range of whole transcriptome - Google Patents

Abstract

Disclosed is a method and kit for detecting an RNA N⁶-methyladenine modification at a single-base resolution in the range of a whole transcriptome. According to the method, on the basis of an N⁶-allyl label of in-vivo ribonucleic acid (RNA) adenine and a chemical treatment, base mutation of the in-vivo RNA adenine in the process of reverse transcription into DNA is induced, and a mutation site is then recognized by means of nucleic acid sequencing, so that an a⁶A site is obtained, wherein the site is a site originally modified by m⁶A in cell RNA. By means of the method, the specific label of N⁶-allyladenine in a cell is achieved for the first time, and the label can not only be used for replacing an N⁶-methyladine site in the cell, but also can be positioned by means of mutation sequencing.

Description

Method and kit for detecting RNA N6-methyladenine modification with single base resolution in full transcriptome range Technical field

The present invention belongs to the field of gene sequencing, and particularly to a full range of single-base resolution transcriptome detecting RNA N ⁶ - methyl adenine modification methods and kits.

Background technique

RNA is not only composed of the four bases of cytosine, thymine, guanine, and adenine. N ⁶ -Methyladenine (m ⁶ A) is an extremely important modified base on RNA, which plays a variety of biological functions in biological processes, such as regulating gene expression. Among them, modification identification and sequencing methods are prerequisites for studying its biological significance.

Because the physicochemical properties of methyl-modified adenine are close to those of ordinary adenine, it cannot be detected directly by the existing first-generation or second-generation sequencing technology. At present, based on m ⁶ A antibody immunoprecipitation sequencing technology, which transcripts and genomes contain m ⁶ A modifications can be obtained, but the resolution is only limited to the range of 100 to 200 bases, and it is impossible to distinguish which A is methylated or not. The A to distinguish whether a single A or a cluster is methylated. In order to improve the resolution, the method of immunoprecipitation after antibody/RNA light cross-linking can also be used, that is, the antibody and the ^{photoactive uracil or thio homologue of the m 6} A neighbor on the RNA are cross-linked. The reverse transcription process leads to cross-linking. Uracil site mutation or termination near the cross-linking position, and then by analyzing the mutation or termination information to indirectly identify the A near the uracil as the m ⁶ A site. Although the resolution of this method has been improved, its position identification is indirect, and m ⁶ A clusters cannot be distinguished.

From the above analysis, it can be seen that the existing ^{high-throughput analysis methods of m 6} A sites on RNA have not achieved major breakthroughs. The main reason is that the current antibody immunoprecipitation enrichment technology is always based on antibody-enriched m ⁶ A sequence fragments. Or cross-linking between the antibody and the ^{photoactive uracil or thio homologue on the m 6} A sequence fragment to indirectly recognize the m ⁶ A site, instead of directly identifying its site by mutation of the ^{m 6 A site} .

The inventor hopes to use the recognizable modification group to introduce the modified group into the cell metabolism by grafting the modification group to the amino acid, replace ^{the methylation modification of m 6} A, and then use the mutation sequencing method. In this way, the site of the modified group is identified, so as to achieve ^{the purpose of identifying the m 6} A site. However, under current culture conditions, amino acids with modified groups and normal amino acids are at a disadvantage in the competition of cell metabolism and cannot be introduced into cells.

Therefore, it is urgent to develop a culture condition suitable for introducing modified amino acids into cells.

Summary of the invention

In view of the shortcomings in the prior art, the purpose of the present invention is to provide a method and kit for ^{detecting RNA N 6 -methyladenine modification with single base resolution in the whole transcriptome range.}

The present invention adopts the following technical solutions:

^{A method for detecting RNA N 6} -methyladenine modification with single-base resolution in the whole transcriptome range, including the following steps:

(1) Allyl-L-seleno/thiohomocysteine participates in cell metabolism and labeling nucleic acid adenine: use methionine analogue allyl-L-seleno/thiohomocysteine Acid-treated cells, cells undergo natural metabolism, and under the action of a series of enzymes, allyl groups can be introduced into the specific adenine N ⁶ position of RNA in the cell to form N ⁶ -allyl adenine (a ⁶ A), the site should be a natural methylation modification, that is, N ⁶ -methyladenine (m ⁶ A);

(2) ^{Enrichment of RNA containing a 6} A modification: extract the total cell RNA, and then further extract the cell mRNA, and then fragment the whole transcriptome RNA of the cell into 100-300 nt fragmented RNA, and use the antibody to bind a ⁶ A , With the help of immunoprecipitation method, enrich a ⁶ A modified RNA, and use eluent to elute the a ⁶ A modified RNA from the antibody, and purify the eluted RNA;

(3) Iodine addition and cyclization of N ^{6 -allyl adenine on RNA a 6 A: A 6} A undergoes iodine addition reaction, and then the N ¹ , N of ^{a 6 A is induced under alkaline conditions. The 6} position forms a circular structure to shield the complementary pairing of bases;

(4) Reverse transcription mutation and sequencing identification of circularized RNA: Add HIV reverse transcriptase to the circularized structure obtained in step (3), and N ¹ , N ⁶ cyclized adenine on RNA acts on HIV reverse transcriptase in vitro In the process of reverse transcription into DNA, an error occurs in the introduction of complementary bases. Nucleic acid sequencing is used to identify the mutation site, and then obtain the a ⁶ A site, which is the original m ⁶ A modified site in the cellular RNA. point.

In the above technical solution, further, in step (1), the preparation method of allyl-L-seleno/thiohomocysteine is: under the protection of nitrogen, first use selenium powder with a molar ratio of 1:1 Se and sodium borohydride NaBH _{4 are used as raw materials, ethanol is used as the solvent, and Na 2} Se _{2 is} prepared by heating and refluxing at 80°C for 6 to 24 hours, and then combining it with selenium powder with a molar amount of one-half to one-third (S )-(+)-2-amino-4-bromobutyric acid hydrobromide (compound 1) was heated and refluxed at 80°C for 6-24 hours, the reaction was terminated with acid, the insoluble matter was removed by filtration, and the by-products were washed away with ether , And then adjust the pH to neutral to obtain selenohomocysteine (compound 2). After that, add sodium borohydride with 1 to 2 times the molar amount of selenium powder to reduce the diselenyl bond, and then under alkaline conditions (molar amount Sodium bicarbonate/sodium carbonate (0.5～2 times of selenium powder) and allyl bromide (0.5～1.5 times of selenium powder/sulfur powder) are reacted at room temperature for 6～24h, and olefin can be obtained by HPLC analysis and purification. Propyl-L-selenohomocysteine (compound 3); using sulfur as a raw material, thiohomocysteine (compound 4) and allyl-L-thiohomocysteine (compound 4) can be prepared Compound 5).

The cells can be, but are not limited to, common mammalian cells, mammalian cancer cells, mammalian stem cells, bacteria, virus host cells, and cells derived from various types of tissues and organs.

Further, the specific method of cell treatment in step (1) is to replace the normal medium of the cell culture system with a medium lacking methionine, and then add 10% FBS, 1% 100x penicillin-streptomycin double On the basis of resistance, add cysteine (0.1～2mM) for 0.5h, then add allyl-L-seleno/thiohomocysteine (0.1～2mM) and continue to incubate for 12-24h After that, the m ⁶ A modification on the RNA can be replaced with a ⁶ A modification. Media lacking methionine can be purchased directly.

Further, in step (2), the whole transcriptome RNA fragmentation treatment of the sample cell is realized by ^{heating Zn 2+} at 70° C. for 4-10 min.

Further, in step (2), ^{the antibody used to bind a 6} A is an antibody of N ⁶ -prenyladenosine, and 10 μg of antibody is used to enrich 1-20 μg of fragmented RNA. Binding antibody, the antibody binds to the fragmented RNA of the whole transcriptome of the above-mentioned sample cell.

Further, the eluent in step (2) is N ⁶ -allyl adenosine triphosphate (a ⁶ ATP), and 5-10 mM a ⁶ ATP is used to compete for washing from N ^{6 -isopentenyl adenosine antibody.} The fragmented RNA of the whole transcriptome of the above-mentioned cells is removed, and the resulting RNA is purified by ethanol or isopropanol precipitation.

Furthermore, in step (3), iodine (0.1-0.5M iodine element dissolved in 0.2-1M potassium iodide) ^{undergoes an iodine addition reaction with the allyl group on a 6} A, and after removing the excess iodine (0.1-0.5M thio Sodium sulfate), under alkaline conditions (0.1-0.5M sodium carbonate, pH=9-10), its N ¹ and N ⁶ positions spontaneously close the ring, so that the normal hydrogen bond pairing of adenine is shielded.

Further, the method of mutation sequencing in step (4) is: i) After reverse transcription of the above-mentioned allyl-L-seleno/thiohomocysteine cell metabolism marker using HIV reverse transcriptase, the antibody is enriched with iodine Add circularized RNA; ii) Use RNA library preparation technology combined with high-throughput sequencing to identify mutation sites in the whole transcriptome to obtain a single-base resolution m ⁶ A site distribution in the whole transcriptome, or use PCR Identify and verify mutation sites on specific transcripts with TA-cloning technology.

Further, the method of the present invention may also have the following characteristics: the amino acids assisted by cell metabolism and in vitro enzymes include but are not limited to allyl-L-selenohomocysteine (allyl-L-selenohomocysteine), allyl -L-thiohomocysteine (allyl-L-homocysteine) and its derivatives and analogues; N ⁶ -allyl adenine nucleoside antibodies include but are not limited to N ⁶ -isopentenyl adenosine Antibodies of its derivatives and analogs; reverse transcription mutant reverse transcriptases include but are not limited to HIV reverse transcriptase.

The method for culturing cells in the above step (1) is to add a methionine derivative and cysteine to a medium lacking methionine, wherein the methionine derivative includes, but is not limited to, allyl-L-selenohomocysteine , Allyl-L-homocysteine and its derivatives and analogs; Cysteine includes but is not limited to the downstream metabolites of methionine in cell metabolism. The chemical structural formulas of allyl-L-selenohomocysteine and allyl-L-homocysteine are as follows

In the above step (1), the method for extracting RNA includes commonly used purification methods or commercial purification kits. Purification methods include, but are not limited to: using TRIzol ^TM Reagent, chloroform-phenol extraction, Proteinase K digestion, silica gel membrane spin column method, magnetic bead method, ethanol, isopropanol precipitation and other techniques or a combination of one or more. Purification kits include but are not limited to: GeneElute ^™ mRNA Miniprep Kit, RNeasy ^™ Mini Kit (Qiagen), RNA Clean & Concentrator (Zymo).

In the above step (2), RNA fragmentation includes but is not limited to RNA fragmentation by metal ion method and RNA fragmentation by ultrasonic disruption; antibody enrichment includes but is not limited to the method of ProteinA/ProteinG beads; elution includes but is not limited to N ⁶ -allyl adenosine monophosphate, a ⁶ ATP competitive elution, TRIzol Reagent extraction elution. The chemical structure of a ⁶ ATP and its synthesis steps are as follows: under the protection of nitrogen, ethanol is the solvent, 6-chloropurine nucleoside (compound 6), allylamine, and triethylamine are in a molar ratio of 1:3:3 in 80 After heating and refluxing at ℃ for 3-6 hours, the product obtained was evaporated in vacuum and then precipitated with ether, filtered to remove insoluble matter, and then recrystallized from methanol to obtain N ⁶ -allyl adenosine (compound 7). Afterwards, under the protection of nitrogen, add N ⁶ -allyl adenosine (0.2 mmol) and phosphorus oxychloride (POCl ₃ , 0.26 mmol) to 0.5 mL of anhydrous trimethyl phosphate (MeO) ₃ PO, and react at 0°C 1.5h, then add tributylammonium pyrophosphate (TBAPP, 1mmol) dissolved in 2mL of anhydrous N,N-dimethylformamide (DMF) into the above reaction system, react at 0℃ for 20min, and further at room temperature After reacting for 5 minutes, the reaction was terminated with 2 mL of triethylammonium bicarbonate (TEAB, 1 mol/L), and analyzed and purified by high performance liquid chromatography (HPLC) to obtain a ⁶ ATP (compound 8). The amount of each reactant can be adjusted in proportion.

In the above step (3), methods for iodine addition include but are not limited to potassium iodide solution of iodine (0.125M I ₂ , 0.25M KI), and methods for removing excess iodine include but are not limited to sodium thiosulfate treatment (0.2M Na ₂ S ₂ O ₃ ), methods for inducing cyclization under alkaline conditions include but are not limited to sodium carbonate, sodium bicarbonate solution (0.1M Na ₂ CO ₃ , pH=9.5) treatment, the reaction formula is as follows:

In the above step (4), the method of reverse transcription includes but is not limited to the method of HIV reverse transcriptase (Recombinant HIV reverse transcriptase enzyme) treatment.

In the above step (4), the sequencing method includes, but is not limited to, high-throughput sequencing for library construction, and low-throughput sequencing based on TA-cloning. Among them, the PCR enzymes in the TA-cloning method include but are not limited to KOD-FX DNA polymerase; the library building methods include, but are not limited to, illuminated stranded library building, NEB small RNA library building, eCLIP and improved library building methods, etc.

In the above steps (1)-(4), the nucleic acid purification steps after each reaction can use common purification methods or commercial purification kits. Purification methods include, but are not limited to: one or more combinations of silica gel membrane spin column method, magnetic bead method, ethanol, isopropanol precipitation and other technologies. Purification kits include but are not limited to: AmpureXP beads,

PCR purification Kit (Qiagen), RNA Clean&Concentrator (Zymo), DNA Clean&Concentrator (Zymo).

The present invention also provides a kit for detecting RNA N ⁶ -methyladenine modification with single-base resolution in the whole transcriptome range, including allyl-L-seleno/thiohomocysteine, cysteine Amino acid, N ⁶ -allyl adenosine triphosphate, RNA fragmentation solution containing divalent zinc ion, N ⁶ -isopentenyl adenosine antibody, 0.125M iodine potassium iodide solution, 0.2M sodium thiosulfate solution, 0.1M Sodium carbonate solution (pH=9.5), HIV reverse transcriptase, HIV reverse transcription reaction solution, Tris-HCl, RNase inhibitor, sequencing adapter, sequencing primer.

In the present invention:

(1) Iodine-induced double bond addition After being added to the double bond, iodine has a certain degree of leaving, and its adjacent carbon atoms can electrophilically attack atoms with higher electron cloud density. For adenine, The nitrogen atom on the purine ring has the characteristic of higher electron cloud density, especially the nitrogen atom at position 1. Through the designed N ⁶ -allyl adenine nucleoside, it is found that under the conditions of iodine addition and alkaline conditions, the allyl iodide at the N6 position can induce the carbon atom to electrophilically attack the adjacent position N1 after the iodine atom leaves. , Forming N1 and N6 ring-closing reactions, shielding the hydrogen bonds originally used for base complementary pairing at these two positions, thereby triggering mutations in the process of reverse transcription, and identifying N ⁶ -allyl adenine by means of sequencing. The purpose of the site;

(2) In order to apply the above-mentioned mutation sequencing method to ^{the identification of m 6} A site, a method of cell metabolism was designed, using the methyl metabolic pathway modified by RNA methylation in the cell to replace the methyl group with the allyl group. Therefore, the m ⁶ A can be replaced with N ⁶ -allyl adenine, and the N ⁶ -allyl adenine site can be identified by mutation sequencing to achieve the purpose of ^{detecting the m 6} A site with single base resolution. The metabolic pathways of RNA methylation modification in the cell are methionine, S-adenosylmethionine, and N ⁶ -methyladenine, which remove the methionine that exists in the cell itself, and then use it in the culture conditions. Allyl-L-seleno/thiohomocysteine replaced methionine and successfully modified the allyl group to the RNA of the whole transcriptome of the cell. ^{Since the modification efficiency of N 6} -allyl adenine nucleoside is identified by quantitative mass spectrometry is low, the present invention finds ^{an antibody that can specifically enrich N 6} -allyl adenine nucleoside, and uses the technique of antibody immunoprecipitation. Enrich ^{the whole transcriptome RNA modified by N 6} -allyl adenine nucleoside, and then follow the above-mentioned iodine addition and cyclization method, use mutation sequencing, and identify the whole transcriptome using both high-throughput and low-throughput approaches Most of the single-base m ⁶ A sites in the range greatly promote the biological function research ^{of m 6 A modification.}

The beneficial effects of the present invention are:

The method of the present invention is based on the chemical labeling of nucleic acid adenine in vivo and inducing mutations. Compared with the existing ^{gene sequencing technology applied to m 6} A detection, the mutation site can be accurate to the single-base resolution, which improves the current universal It is a direct high-throughput single-base identification method based on m ⁶ A antibody immunoprecipitation and massively parallel sequencing to detect ^{the accuracy of m 6 A sites.}

^{The present invention realizes the N 6} -allyl labeling of adenine in the inner ribonucleic acid of the cell for the first time, which provides the possibility to identify its site by means of mutation of the ^{m 6 A site.}

Since ^{the modification efficiency of N 6} -allyl adenosine is low, the present invention uses N ⁶ -prenyl adenosine antibodies to specifically enrich N ⁶ -allyl adenosine antibodies. Because there is currently no N ⁶ -allyl adenosine specific antibody, after analysis and screening of some commercial antibodies, it was found that the N ⁶ -isopentenyl adenosine antibody is against N ⁶ -allyl gland Purine nucleoside has a certain specificity, the antibody ^{has good binding ability with N 6} -allyl adenosine nucleoside, and commercial antibodies are easier to obtain, which greatly improves the practicability of the method.

The present invention uses a ⁶ ATP as the eluent, because a ⁶ ATP has the ^{same main structure as the a 6} A modification on RNA, and the triphosphate increases its water solubility and has stronger binding ability with antibodies, making it modified ^{with a 6 A} In the process of binding to the antibody, the RNA of α is at a competitive advantage, so the α ⁶ A modified RNA can be eluted by competing with the antibody. Therefore, compared with the common RNA extraction method for eluting RNA, this method makes a ⁶ A modified RNA and antibody almost completely separated, which greatly improves the yield of eluted RNA.

The present invention uses HIV reverse transcriptase to perform reverse transcription processing on iodine addition and inducing circularization of RNA, because the inventors have found that HIV reverse transcriptase has a shielded circular structure for the hydrogen bond of adenine for base complementary pairing. With stronger recognition ability, the inventor selected many commercial reverse transcriptases, including HIV reverse transcriptase (Reverse Transcriptase Recombinant HIV, Worthington Biochemical Corporation), M-MLV reverse transcriptase (PROMEGA, M170A), AMV reverse transcriptase ( PROMEGA, M510F), RevertAid reverse transcriptase (Thermofisher, EP0441), SuperScript Ⅱ reverse transcriptase (Invitrogen, 18064022), SuperScript Ⅲ reverse transcriptase (Invitrogen, 18080093), etc., finally found that HIV reverse transcriptase in a ⁶ A iodine Mutations occur at the corresponding sites during the reverse transcription of the addition circular RNA.

Based on mutation sequencing, the present invention can be applied to a variety of analysis methods based on gene sequencing, such as ^{detection of m 6} A modification sites ^{on various types of nucleic acids, and cells based on N 6} -allyl adenosine RNA dynamic sequencing, etc.

Description of the drawings

Figure 1 is a schematic diagram of the modification method of the present invention;

^{Figure 2 shows the content of N 6} -allyl adenine nucleoside in mRNA after allyl-L-seleno/thiohomocysteine is introduced into HeLa cells for metabolism;

Figure 3 is an RNA band obtained by fragmentation treatment of sample cell mRNA by divalent zinc ions;

Figure 4 is ^{a dot-hybrid diagram of N 6} -prenyladenosine antibody and normal A, m ⁶ A modified RNA, a ⁶ A modified RNA, which shows the binding ability of the corresponding RNA and antibody, and methylene blue shows the amount of RNA loaded ；

^{Figure 5 shows the N 6} -allyl adenine nucleoside content before and after mRNA enrichment in HeLa and H2.35 cells by antibody immunoprecipitation method;

Figure 6 shows ^{the results of gene sequencing of a 6} A modified RNA in HeLa cells before and after iodine addition-induced circularization;

Figure 7 is the conservative sequence ^{of the m 6} A site on the transcriptome obtained by high-throughput sequencing of HeLa cells and H2.35 cells;

^{Figure 8 is the m 6} A site on the transcriptome obtained by high-throughput sequencing of HeLa cells, taking three mRNA gene sequences as examples;

^{Figure 9 is the m 6} A site on the transcriptome obtained by high-throughput sequencing of H2.35 cells, taking four mRNA gene sequences as examples;

^{Figure 10 is the m 6} A site on the transcriptome obtained by low-throughput sequencing of HeLa cells, taking three mRNA gene sequences as examples;

Figure 11 is the high resolution mass spectrum of allyl-L-selenohomocysteine;

^{Figure 12 is the 1} H NMR spectrum of allyl-L-selenohomocysteine;

^{Figure 13 is the 13} C NMR spectrum of allyl-L-selenohomocysteine;

Figure 14 is the high resolution mass spectrum of allyl-L-homocysteine;

Figure 15 is a high resolution mass spectrum ^{of a 6 ATP.}

detailed description

The following describes the present invention in detail with reference to the drawings and specific embodiments, but they should not be understood as limiting the scope of protection of the present invention.

As shown in Figure 4, three types of RNA were obtained by in vitro transcription, namely normal RNA (A-RNA), A was replaced by m ⁶ A RNA (m ⁶ A-RNA), and A was replaced by a ⁶ A RNA (a ⁶ A-RNA), it can be seen from the figure that only a ⁶ A-RNA (compared to A-RNA and m ⁶ A-RNA) has a relatively specific binding ability with antibodies, which can be derived from cellular mRNA The normal A sequence and the m ⁶ A modified sequence ^{identify the a 6} A modified mRNA to provide an enrichment function. As a control, Methylene blue shows the loading amount of the corresponding RNA, which proves that the same amount of RNA is under a ⁶ The specific binding ability of A-RNA and antibody.

In the present invention, after obtaining the immunoprecipitated circularized and uncircularized products, the fragmented RNA is subjected to HIV reverse transcription and then connected to a sequencing adapter, and the sequencing primers are used for PCR amplification to construct a high-throughput sequencing library. The high-throughput library is preferably illumina Truseq stranded mRNA LT kit. Sequencing the constructed sequencing library can obtain sequencing data. The sequencing is preferably next-generation sequencing, more preferably Illumina paired-end sequencing, and the read length of sequencing is preferably 150 bp.

After obtaining the sequencing data, analyze the mutation rate of the circularized and uncyclized samples at the adenylate sites. If the mutation rate of the circularized sample relative to the uncircularized sample is greater than 3, and in the a ⁶ A antibody-rich region, It is considered that this site is the m ⁶ A site. The analysis methods are: quality control of sequencing data, removal of adapter sequences and low-quality bases, alignment of the data with low-quality and adapters removed to the genome sequence and enrichment statistics, and comparison of data with low-quality and adapters removed Go to the transcriptome sequence and perform mutation statistics; specifically include the following steps: quality control of the original sequencing data through fastqc default parameters, and fastp software to remove linker sequences and sequences less than 25 bases (fastp -f 10 -F 10- x --detect_adapter_for_pe-l 25 –i Raw-R1.fq –I Raw-R2.fq –o Clean-R1.fq –O Clean-R2.fq), the obtained sequence is again used for quality control with the default parameters of the fastqc software, Then use hisat2 default parameters to align to the transcriptome sequence, use samtools to convert the aligned SAM file into a BAM file, and use samtools rmdup to remove repetitive sequences. Finally, use samtools mplieup to count the mutation information in the BAM file to compare circularization and uncirculation For the mutation rate of the chemical sample at the same site, if the fold change is greater than 3, it is considered as the m ⁶ A site.

After obtaining the immunoprecipitated circularized and uncircularized products, PCR and TA-cloning technology can also be used to perform low-throughput sequencing of a certain transcriptome to identify mutation sites. The DNA polymerase for PCR is preferably KOD-FX DNA polymerase, and the plasmid vector for connecting the PCR product is preferably a T vector. For example, as shown in Figure 6, the results of a certain a ⁶ A-labeled RNA through PCR and TA-cloning technology low-throughput sequencing, where X = a ⁶ A means that the site is determined to have a ⁶ A modification, X = cyc- A indicates that the ^{modified site of a 6} A has undergone iodine addition and induced cyclization. The results show that HIV reverse transcriptase has a mutation at the X=cyc-A site, but no mutation at the X=a ⁶ A site , To prove the necessity of iodine addition and induction of cyclization and the necessity of HIV reverse transcriptase. Comparing the mutation rate of the circularized and uncyclized samples at the same site, it is found that the fold change is greater than 3, which confirms that the m ⁶ A site can be accurately detected by the method of the present invention.

Figure 11 shows the high resolution mass spectrum of allyl-L-selenohomocysteine prepared by the present invention (m/z is 224.0183, [M+H] ⁺ theoretical calculation is 224.0184); Figure 12 shows the ¹ H nuclear magnetic resonance spectrum, ¹ H NMR ( 500MHz, D ₂ O), Figure 13 ^{shows the 13} C nuclear magnetic resonance spectrum, ¹³ C NMR (126MHz, D ₂ O); combined with Figures 11-13, it can be seen that allyl-L-selenohomocysteine was successfully prepared. Similarly, in conjunction with Figure 14 (C ₇ H ₁₃ NO ₂ S, m/z is 176.0729, [M+H] ⁺ theoretical calculation is 176.0740), it can be seen that allyl-L-homocysteine was successfully prepared according to this method.

A ⁶ ATP 15 of the present invention is prepared as shown in the relevant characterization data (high resolution mass _{spectrum, C 13 H 20 N 5 O} 13 P 3, m / z was 546.0192, [MH] ^- theoretical 546.0198), found successfully prepared a ⁶ ATP.

^{Example 1 Detection of m 6} A modification sites on transcriptome mRNA in HeLa cells

1. HeLa cell culture, allyl labeling and mRNA extraction

(1) After HeLa cells are cultured to a fullness of 80% under normal culture conditions, aspirate the medium and wash away the residual medium with PBS;

(2) Add 10% FBS, 1% 100x penicillin-streptomycin double antibody, and 1 mM cysteine to the medium without methionine, and culture the above HeLa cells in this medium for 30 minutes , To remove the residual methionine in the cell;

(3) After removing the methionine, add 1 mM allyl-L-selenohomocysteine/allyl-L-homocysteine to the medium for culturing the HeLa cells, and continue culturing for 16 hours;

(4) After cell culture is completed, aspirate the medium, wash away the residual medium with PBS, and then add TRIzol ^TM Reagent to the petri dish to cover the bottom of the petri dish, eluting all the adherent HeLa cells into the solution, and transfer to Lysis in a centrifuge tube at room temperature for 5 minutes;

(5) For every 1mL of TRIzol ^TM Reagent, add 0.2mL of chloroform to the above centrifuge tube, shake the centrifuge tube vigorously for 15 seconds, extract at room temperature for 3 minutes, centrifuge at 4℃, RCF 12000g for 15 minutes, layer the solution, and the supernatant is RNA , The white precipitate in the middle is DNA, and the red liquid in the lower layer is protein;

(6) Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of isopropanol, incubate on ice for 10 minutes, and centrifuge at 4°C RCF 15000g for 15 minutes to obtain a total RNA white precipitate;

(7) Remove the supernatant, leaving the total RNA white particles, wash the pellet with 1mL 75% ethanol/1mL TRIzol, centrifuge at 4℃RCF 15000g for 15min, remove the supernatant again, air dry for 3min, use 250μL of RNase-free Dissolve total RNA in water, heat at 70°C for 10 minutes to fully dissolve total RNA, use BioDrop to determine the RNA concentration to be 1000ng/μL;

(8) Use GenElute mRNA MiniprepKit to further extract mRNA. Take 250μL of total RNA in a 1.5mL centrifuge tube, add 250μL of 2xBinding Solution, gently shake the centrifuge tube to mix the solution;

(9) Add 20μL of evenly dispersed oligo(dT)beads and shake to mix the system thoroughly. Heat at 70°C for 3min to denature the RNA, then incubate at room temperature for 10min to fully combine the oligo dT and RNA. Centrifuge the mixed system at RCF 15000g for 2min Obtain oligo dT beads with mRNA, carefully remove the supernatant, and leave about 50 μL of the solution to prevent the loss of beads;

(10) Add 500 μL of Wash Solution to the centrifuge tube to fully suspend the oligo dT by pipetting, transfer to the GenElute centrifugal filter column/collection tube, centrifuge at 15000g RCF for 2 minutes, and discard the liquid in the collection tube;

(11) Add 500μL of Wash Solution to the filter column again, centrifuge at 15000g RCF for 2min, and discard the liquid in the collection tube;

(12) Finally, transfer the filter column to a new collection tube, draw 50 μL of Elution Solution preheated at 70°C and add it to the center of the filter membrane of the centrifugal filter column, fully contact the mRNA complex in the column hole, and incubate at 70°C Centrifuge at RCF15000g for 1min for 5min, and get the mRNA solution in the collection tube. Use BioDrop to determine the mRNA concentration to be 200ng/μL. In addition, you can also pipette another 50μL of Elution Solution at 70°C to repeat the above elution process, so that the mRNA in the pores of the filter column is fully dissolved, and the obtained mRNA is stored at -80°C.

2. RNA enrichment with allyl modification at the ⁶ -position of adenine N

(1) Mix 200 μL of the above RNA sample, 20 μL of 3M sodium acetate solution, 220 μL of isopropanol, and 2 μL of glycogen, then pipette and mix well. After precipitation overnight, centrifuge at 4°C at 15000 rpm for 45 min, wash the precipitate with 440 μL of 80% ethanol, 15000 rpm Centrifuge at 4°C for 15 minutes, remove the supernatant again, air dry for 3 minutes, and dissolve RNA with 70μL of RNase-free water to a concentration of 550ng/μL;

(2) Mix 45μL of the above RNA sample (450～650ng/μL) with 5μL of Zn ²⁺ fragmentation buffer (10x Fragment buffer), pipette and mix, heat at 70℃ for 7min, then add 10μL of 0.5M EDTA solution to terminate the fragmentation The composition of 10x Fragment buffer is shown in Table 1;

Table 1 10x Fragment buffer

(3) Mix the above 60μL solution with 6μL 3M sodium acetate solution, 66μL isopropanol, 1μL glycogen and mix by pipetting. After precipitation overnight, centrifuge at 4℃ at 15000rpm for 45min, wash the precipitate with 132μL of 80% ethanol at 15000rpm Centrifuge at 4°C for 15 minutes, remove the supernatant again, air dry for 3 minutes, and dissolve RNA with 200 μL of RNase-free water to a concentration of 100 ng/μL. The RNA fragments obtained are shown in Figure 3, and the fragment length is 100-300 nt;

(4) Mix 50μL of the above fragmented RNA with 80μL (5x IP buffer, 2μL RNA RNase inhibiter, 1μL N ⁶ -isopentenyl adenosine antibody, 267μL RNase-free water, pipetting and mixing, rotating at 4°C for 4h , Make N ⁶ -allyl adenosine modified RNA and N ⁶ -prenyl adenosine antibody fully bind, 5x IP buffer components are shown in Table 2;

Table 2 5x IP buffer

(5) Separate the magnetic beads with 40μL ProteinA magnetic beads on the magnetic stand, wash with 200μL 1x IP buffer three times, aspirate the remaining clear liquid, and then mix with 80μL 5x IP buffer, 10μL 20mg/μLBSA, 310μL RNase-free water, and pipette Mix, rotate and shake at 4°C for 2 hours to prevent non-specific adsorption of magnetic beads when binding to antibodies. The composition of the 1x IP buffer is shown in Table 3;

Table 3 1x IP buffer

(6) After the RNA and the antibody are fully bound, separate the magnetic beads on the magnetic stand with the mixture of ProteinA magnetic beads and BSA, wash them with 200μL 1x IP buffer three times, aspirate the remaining clear liquid, and then combine the ProteinA magnetic beads with RNA- After mixing the antibody mixture, mix by pipetting, and incubate for 2 hours with rotation and mixing at 4°C, so that the RNA-binding antibody binds to the ProteinA on the magnetic beads;

(7) Put the above mixed solution on the magnetic stand to separate the magnetic beads, wash with 200μL 1x IP buffer three times, aspirate the remaining clear liquid, ^{mix with 100μL a 6} ATP elution buffer (Elution buffer) and ProteinA magnetic beads, then pipette to mix. Evenly, rotate and shake at 4°C for 3 hours, ^{and elute the N 6} -allyl adenine nucleoside modified RNA from the antibody bound to the ProteinA magnetic beads into the solution. The Elution buffer composition is shown in Table 4;

Table 4 Elution buffer

(8) Mix 4 50μL fragmented RNA (100ng/μL) elution products after antibody immunoprecipitation to obtain 400μL solution, mix with 40μL 3M sodium acetate solution, 440μL isopropanol, 2μL glycogen and then mix by pipetting and mixing. After precipitation overnight, centrifuge at 15000rpm at 4°C for 45min, wash the pellet with 880μL of 80% ethanol, centrifuge at 15000rpm at 4°C for 15min, remove the supernatant again, air dry for 3min, and dissolve RNA with 20μL of RNase-free water to obtain a concentration of 15ng/μL. After RNA hydrolysis obtained for individual nucleosides, after quantitative liquid chromatography mass, to give a ⁶ A longitudinal IP RNA content is shown in FIG. 5, HeLa Input IP prior to the mRNA in HeLa cells in a ⁶ A content, HeLa IP is a ⁶ a content after IP, H2.35Input front H2.35 IP cells in mice in the mRNA content of a ⁶ a, H2.35IP as a ⁶ a content IP, the results show that, indeed a ⁶ a IP modified mRNA was enriched.

3. Iodine addition reaction and cyclization of ^{N 6 -allyl adenine of RNA}

(1) Transfer 20μL of the above antibody-enriched RNA (15ng/μL) to a PCR tube, dilute to 26μL with RNase-free water, add 4μL of 0.125M iodine solution (dissolved in 0.25M potassium iodide), the solution becomes Brown, treated at 37°C for 30min;

(2) Transfer the above brown solution to a new PCR tube, add 4μL of 0.2M sodium thiosulfate until the solution is colorless, then add 6μL of 0.1M sodium carbonate (pH=9.5), and treat at 37°C for 30min;

(3) Mix 40μL of the above solution with 40μL of isopropanol and 1μL of glycogen and mix by pipetting. After precipitation overnight, centrifuge at 15000rpm at 4℃ for 45min, wash the precipitate with 880μL of 80% ethanol, and centrifuge at 15000rpm at 4℃ for 15min , Remove the supernatant again, air-dry for 3 minutes, and dissolve the RNA with 15 μL of RNase-free water to obtain a concentration of 10 ng/μL.

4. The RNA fragments obtained without immunoprecipitation and immunoprecipitation (including circularization treatment and non-circularization treatment) are used to construct a library using the Illumina mRNA library construction kit;

(1) Configure the reverse transcription system according to the reaction system in Table 5;

Table 5 Reverse transcription reaction system for mRNA library construction

(2) After mixing, run the program in the PCR machine as shown in Table 6;

Table 6 Reverse transcription reaction PCR running program for mRNA library construction

(3) Add 5μL resuspension buffer, 20μL Second Strand Marking Master Mix to the above system, mix and incubate at 16°C for 1 hour;

(4) Purify the double-stranded DNA fragments obtained above: add 100 μL of AMPure XP beads that have been returned to room temperature in advance to the reaction system, mix by pipetting 6-10 times, incubate at room temperature for 15 minutes, separate with a magnetic stand, discard the supernatant, and use 80% Wash the beads twice with ethanol for 30 seconds each time, let stand for 5-10 minutes to volatilize the ethanol, eluate the double-stranded DNA fragments with 17.5μL resuspension buffer, incubate for 2min at room temperature and separate on a magnetic stand, take 15μL of supernatant for the next step;

(5) The double-stranded DNA obtained above is added with an adenylate tail according to the reaction system configuration in Table 7;

Table 7 Reaction system for adding adenylate tail to mRNA library building

(6) After the above system is pipetted and mixed evenly, the running program in the PCR machine is shown in Table 8;

Table 8 PCR running program for mRNA library construction with adenylate tail added

(7) Press the list 9 to configure the reaction system to add connectors;

Table 9 Reaction system for adding linker to mRNA library configuration

(8) Incubate the above reaction system at 30°C for 10 minutes;

(9) Add 5 μL of the connection termination mixture to the above reaction, and mix by pipetting;

(10) Purify the DNA fragments connected to the linker sequence: Add 42.5 μL of AMPure XP beads that have been restored to room temperature in advance to the reaction system, mix by pipetting 6-10 times, incubate at room temperature for 15 minutes, separate with a magnetic stand and discard the supernatant. Wash the beads twice with 80% ethanol, 30 seconds each time, let stand for 5-10 minutes to volatilize the ethanol, eluate the double-stranded DNA fragments with 52.5μL resuspension buffer, incubate for 2min at room temperature and separate on a magnetic stand, take 50μL supernatant, and add 50μL AMPure XP beads, repeat the purification steps, eluted with 22.5μL resuspension buffer, and take 20μL of supernatant for the next step of PCR library amplification;

(11) Configure the library amplification system according to the reaction system in Table 10 below;

Table 10 Amplification reaction system for mRNA library construction

(12) See Table 11 for the running program in the PCR machine after pipetting and mixing the above system;

(13) Purify the above amplified library: add 55μL of AMPure XP beads that have been returned to room temperature in advance to the reaction system, mix by pipetting 6-10 times, incubate at room temperature for 15 minutes, separate with a magnetic stand and discard the supernatant, use 80% Wash the beads twice with ethanol for 30 seconds each time, let stand for 5-10 minutes to volatilize the ethanol, eluate the double-stranded DNA fragments with 22.5μL resuspension buffer, incubate for 2min at room temperature and separate on a magnetic stand, take 20μL of supernatant, of which 1μL is used for Qubit Determine the concentration;

Table 11 The operating program of PCR for mRNA library construction and library amplification

(14) Build a library of the obtained mRNA, use the Illumina X-10 platform to perform paired-end 150 sequencing, and compare the obtained data with the transcriptome of the tested cell-derived species after low-quality filtering and adapter filtering. Click the sequence features before and after the dots to obtain the conservative sequence ^{of the m 6} A site on the transcriptome of HeLa cells and mouse H2.35 cells, which is roughly the same as the conservative sequence obtained by the ^{common m 6 A antibody immunoprecipitation and sequencing technology.} The site identified by this method is the m ⁶ A site (Figure 7).

(15) The high-throughput sequencing results are shown in Figure 8. The mutation sequencing results show that LATS1 (NM_001350339) has m ⁶ A modification at 2970 and 2991, and ZNF445 (NM_181489) has m 6 A modification at 3451 and 3462. m ⁶ A modification, OTUD1 (NM_001145373) has m ⁶ A modification at position 1479.

5. The RNA fragments treated with iodine addition and circularization were subjected to low-throughput sequencing using TA cloning technology to verify the mutation of specific sites on the mRNA of HeLa cells, and to further identify the m ⁶ A site

(1) Take 1μL of the above-mentioned iodine addition and circularized RNA (10ng/μL) into a PCR tube, mix with 1μL 10μM reverse primer (R-primer), 12μL RNase-free water, pipetting and mixing, heat at 65℃ After denaturation for 5 minutes, the R-primer sequence is shown in Table 12;

Table 12 Sequences of F-primer and R-primer

(2) After cooling on ice, add 4μL 5x Reverse Transcription Reaction Buffer (Thermofisher, EP0441), 1μL 10mM dNTP, 1μL 10U/μL HIV Reverse Transcriptase (Worthington Biochemical Corporation), react at 37°C for 1 hour, then heat at 70°C 10min to inactivate reverse transcriptase;

(3) Take 11μL of the above reverse transcription reaction solution to a new PCR tube, use KOD-FX (TOYOBO, KFX-101) enzyme for PCR amplification, add 10μL 2mM dNTP, 1.5μL 10μM F-primer and R-primer, 25μL 2x KOD-FX reaction buffer, and 1μL KOD-FX enzyme, mix, pipette and mix well, F-primer and R-primer sequence are shown in Table 12, PCR reaction program is shown in Table 13;

Table 13 PCR amplification reaction program after reverse transcription

(4) After the PCR reaction is completed, the product DNA is purified with 1.8 times the volume of AMPure XP beads (BECKMAN COULTER, A63881), and the DNA is dissolved in 15 μLRNase-free water to obtain a concentration of 50ng/μL;

(5) Use pUCm-T Vector (Sangon Biotech, B522213) to ligate 1 μL of the above DNA product with T vector, and react at 16°C for 6 hours. The pUCm-T Vector vector ligation system is shown in Table 14;

Table 14 pUCm-T Vector carrier connection system

(6) Place 100 μL of DH5α competent cells on ice to thaw for 5 minutes until the cells are evenly suspended, add 5 μL of the above-mentioned connection solution, tap gently to mix, and place on ice for 25 minutes;

(7) Heat shock in 42°C water bath for 45 seconds, then place on ice for 2 minutes, add 700μL of SOC medium, shake culture at 37°C 220 rpm for 1 hour;

(8) Centrifuge at 5000 rpm for 1 min, aspirate 600 μL of supernatant, and suspend the cells with the remaining medium;

(9) Configure 50mL LB plates containing 50μL IPTG (100mM), 100μL X-gal (20mg/mL), 50μL 1000x ampicillin, 15mL per plate, and evenly spread the above bacterial suspension on the LB plate;

(10) Incubate the plate at 37°C for 1 hour, then invert the culture for 16 hours, screen the white single colonies in the blue and white spots to extract the plasmid for Sanger sequencing to obtain low-throughput sequencing results;

(11) The low-throughput sequencing results are the same as the high-throughput sequencing results. As shown in Figure 10, LATS1 (NM_001350339) has m ⁶ A modification at two positions 2970 and 2991, ZNF445 (NM_181489) has m ⁶ A modification at two positions 3451 and 3462, and OTUD1 (NM_001145373) has m 6 A modification at position 1479. There is m ⁶ A modification.

^{Example 2 Detection of m 6} A modification sites on transcriptome mRNA in mouse H2.35 cells

1. Culture, allyl labeling and mRNA extraction of mouse H2.35 cells

(1) Mouse H2.35 cells are cultured under normal culture conditions (additional 200nM DEX, dexamethasone is required), after culturing to a fullness of 60%, aspirate the medium and wash away the residual medium with PBS;

(2) Add 10% FBS, 1% 100x penicillin-streptomycin double antibody, 200 nM DEX, and 1 mM cysteine to the medium without methionine to remove the above mouse H2.35 cells Incubate in this medium for 30 minutes to remove the residual methionine in the cells;

(3) After removing the methionine, add 1mM allyl-L-selenohomocysteine/allyl-L-homocysteine to the medium for culturing the above-mentioned mouse H2.35 cells, and continue culturing for 16h;

(4)-(12) Same as HeLa cell culture, allyl labeling and mRNA extraction in Example 1.

2-4. Same as steps 2-4 in Example 1.

5. The high-throughput sequencing results are shown in Figure 9. The mutation sequencing results show that Xist (NR_001463) has m ⁶ A modification at two positions 11956 and 11964, and Usp42 (NM_029749) has m ⁶ A modification at 2973 position. Ice1 (NM_144837) has m ⁶ A modification at position 3777, and Eppk1 (NM_144848) has m ⁶ A modification at two positions 2899 and 2924.

6. The RNA fragments processed by iodine addition and circularization were subjected to low-throughput sequencing using TA cloning technology to verify the mutation of specific sites on the HeLa cell mRNA, and to further identify the m ⁶ A site, as in step 5 in Example 1. .

Example 3 Cultivation of HeLa cells and mouse H2.35 cells under normal cell culture conditions

1. Cultivation of HeLa cells

Add 10% FBS, 1% 100x penicillin-streptomycin double antibody to normal cell culture medium. After HeLa cells are cultured in this medium for 16-24 hours, the mRNA of HeLa cells can be extracted, and the m ⁶ A content of HeLa cells can be extracted. As shown by Ctrl in Figure 2.

Cultivate HeLa cells according to the treatment conditions of the allyl labeling method in Example 1, add 1mM allyl-L-selenohomocysteine, allyl-L-homocysteine, or methionine, culture for 16h, and get the m ⁶ A content of mRNA as shown in Figure 2. Shown. In Figure 2 Se-allyl-L-selenohomocysteine/S-allyl-L-homocysteine is Se-allyl-L-selenohomocysteine/S-allyl-L-homocysteine introduced into HeLa cells for metabolism to obtain N ⁶ -allyl adenine in mRNA The content of nucleosides, L-Methionine is the N ^{6 -methyl adenine nucleoside content in the mRNA obtained after methionine is introduced into HeLa cells for metabolism, Ctrl is the N 6} -methyl adenine in the mRNA obtained after normal culture of HeLa cells The content of nucleoside shows that the labeling efficiency of allyl-L-selenohomocysteine is higher than that of allyl-L-homocysteine, but it is much lower than the m ⁶ A level in normal cultured cells, and after 0.5h of removal of intracellular methionine After the treatment, methionine was added again, and the m ⁶ A level of the cells was almost normal, indicating that the treatment of removing methionine in a short period of time did not have an excessive effect on the cell state.

2. Cultivation of mouse H2.35 cells

Add 10% FBS, 1% 100x penicillin-streptomycin double antibody, 200 nM DEX to normal cell culture medium, and culture mouse H2.35 cells in this medium for 16-24 hours to extract their mRNA.

The HeLa cells were cultured according to the treatment conditions of the above-mentioned allyl labeling method to obtain m ⁶ A of each group of mRNA. The result is similar to the above-mentioned HeLa cells. The labeling efficiency of allyl-L-selenohomocysteine is higher than that of allyl-L-homocysteine, and its labeling level is much lower than the m ⁶ A level in normal cultured cells. However, after 0.5h of depleted cells After methionine treatment, methionine was added again, and the m ⁶ A level of the cells was almost normal.

Claims (10)

1. ^{A method for detecting RNA N 6} -methyladenine modification with single-base resolution in the whole transcriptome range, which is characterized in that it comprises the following steps:

   (1) Allyl-L-seleno/thiohomocysteine labeled nucleic acid adenine: treat cells with the methionine analogue allyl-L-seleno/thiohomocysteine, The cell introduces the allyl group into the specific adenine N ⁶ -methyladenine position ^{of RNA in the cell through natural metabolism to form N 6} -allyl adenine a ⁶ A;

   (2) ^{RNA enrichment containing a 6} A modification: extract the total cell RNA, and then further extract the mRNA of the cell, and then fragment the whole transcriptome RNA of the cell into 100-300 nt fragmented RNA, and use the antibody to bind N ⁶ -Allyl adenine nucleoside, with the help of immunoprecipitation method, enrich a ⁶ A modified RNA, and use eluent to elute the a ⁶ A modified RNA from the antibody, and purify the eluted RNA;

   (3) N ⁶ on RNA a ⁶ A - with the addition of allyl iodide adenine ring of treatment: N ⁶ on RNA a ⁶ A - adenine allyl iodide addition reaction occurs, and then in a basic Under conditions, induce the N ¹ and N ⁶ positions of the N ^{6 -allyl adenine on RNA a 6} A to form a circular structure to shield the complementary pairing of bases;

   (4) Reverse transcription mutation and sequencing identification of circularized RNA: Add HIV reverse transcriptase to the circularized structure obtained in step (3). The N ¹ , N ⁶ circularized adenine on RNA acts on HIV reverse transcriptase In the process of reverse transcription into DNA, an error occurs in the introduction of complementary bases. Nucleic acid sequencing is used to identify the mutation site, and then obtain the a ⁶ A site, which is the original m ⁶ A modified site in the cellular RNA. point.
2. ^{The method for detecting RNA N 6} -methyladenine modification with single-base resolution across the entire transcriptome according to claim 1, wherein in step (1), allyl-L-seleno/thio The preparation method of homocysteine is as follows: under the protection of nitrogen, using selenium powder/sulfur powder and sodium borohydride in a molar ratio of 1:1 as raw materials, ethanol as the solvent, heating and refluxing at 80°C for 6-24 hours; then adding The hydrobromide of (S)-(+)-2-amino-4-bromobutyric acid whose molar amount is one-half to one-third of selenium powder/sulfur powder is heated and refluxed at 80℃ for 6-24 hours Add acid to terminate the reaction, filter to remove insolubles, wash away by-products with ether, and adjust the pH to neutral; then add sodium borohydride with a molar amount of selenium powder/sulfur powder 1-2 times to reduce the diselenyl/disulfide bond, Then add sodium bicarbonate/sodium carbonate with a molar amount of 0.5 to 2 times of selenium powder/sulfur powder and allyl bromide with a molar amount of 0.5 to 1.5 times of selenium powder/sulfur powder to react at room temperature for 6 to 24 hours, and pass the high performance liquid chromatography Analyze and purify to obtain allyl-L-seleno/thiohomocysteine.
3. ^{The method for detecting RNA N 6} -methyladenine modification with single-base resolution in the full transcriptome range according to claim 1, wherein in step (1), the cells are ordinary mammalian cells, mammalian cells, and mammalian cells. Host cells for animal cancer cells, mammalian stem cells, bacteria, viruses, or from various types of tissues and organs.
4. ^{The method for detecting RNA N 6} -methyladenine modification with single-base resolution across the entire transcriptome according to claim 1, wherein the specific method for cell processing in step (1) is to use a lack of methionine Cell culture in acid medium, and add 0.1-2mM cysteine for 0.5h, then add 0.1-2mM allyl-L-seleno/thiohomocysteine for 12-24h .
5. ^{The method for detecting RNA N 6} -methyladenine modification with single base resolution in the whole transcriptome range according to claim 1, wherein in step (2), the whole transcriptome RNA of the cell is fragmented by Zn ²⁺ ions are heated at 70°C for 5-10 minutes.
6. ^{The method for detecting RNA N 6} -methyladenine modification with single-base resolution across the entire transcriptome according to claim 1, wherein in step (2), it is used to bind N ⁶ -allyl adenine The nucleoside antibody is N ⁶ -prenyladenosine antibody, and 10 μg antibody is used to enrich 1-20 μg fragmented RNA.
7. ^{The method for detecting RNA N 6} -methyladenine modification with single base resolution in the whole transcriptome range according to claim 1, characterized in that, in step (2), the eluent is N ⁶ -ene Propyl adenosine triphosphate, the concentration is 5-10mM.
8. ^{The method for detecting RNA N 6} -methyladenine modification with single-base resolution in the whole transcriptome range according to claim 1, wherein the step (3) is specifically: adding 0.1-0.5M iodine elemental substance Dissolve in 0.2～1M potassium iodide to obtain the potassium iodide solution of iodine, then make the potassium iodide solution of iodine react with the allyl group of N ^{6 -allyl adenine on RNA a 6} A, and then remove with 0.1～0.5M sodium thiosulfate Excessive iodine; add 0.1～0.5M sodium carbonate and adjust pH=9～10 to induce the N ¹ and N ⁶ positions of N ^{6 -allyl adenine on RNA a 6} A to form a cyclic structure, thereby making adenine Normal hydrogen bond pairing is shielded.
9. ^{The method for detecting RNA N 6} -methyladenine modification with single-base resolution across the entire transcriptome according to claim 1, wherein the step (4) is specifically: i) reverse transcriptase using HIV reverse transcriptase Record the above-mentioned iodine-added circularized RNA; ii) Use RNA library preparation technology combined with high-throughput sequencing to identify mutation sites in the whole transcriptome to obtain the ^{distribution of m 6} A sites with single base resolution in the whole transcriptome, Or use PCR and TA-cloning technology to identify and verify mutation sites on specific transcripts.
10. ^{A kit for detecting RNA N 6} -methyladenine modification with single-base resolution in the whole transcriptome range, characterized in that it includes allyl-L-seleno/thiohomocysteine, cysteine Acid, N ⁶ -allyl adenosine triphosphate, RNA fragmentation solution containing divalent zinc ion, N ⁶ -prenyl adenosine antibody, potassium iodide solution of iodine, sodium thiosulfate solution, sodium carbonate solution, HIV reverse transcription Enzyme, HIV reverse transcription reaction solution, Tris-HCl, RNase inhibitor, sequencing adapter, sequencing primer.

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