Universal library building method for trace RNA modification detection and application of universal library building method in RNA modification detection

Abstract

The invention relates to a general library building method for trace RNA modification detection and application of the general library building method in RNA modification detection. The general library building method comprises the following steps: S1, collecting an RNA sample without ribosomal RNA; s2, repairing the tail end of the RNA sample; s3, taking the RNA chain in the S2 as a template, carrying out reverse transcription to form a first cDNA chain, synthesizing a second cDNA chain, and hydrolyzing the template; s4, taking the second cDNA as a template, and carrying out in-vitro transcription to obtain an RNA chain; s5, taking the RNA chain in the S4 as a template, and carrying out reverse transcription to obtain a cDNA chain; s6, taking the cDNA chain in S5 as a material for constructing an RNA library to obtain the RNA library; wherein S2 is performed on the RNA sample in S1, or S2 is performed after the RNA apparent modification of the RNA sample in S1 is pretreated, or S3 is performed after the RNA apparent modification of the RNA chain in S2 is pretreated. According to the invention, high-throughput detection of a low-initial-quantity RNA sample is realized, and quantitative detection of RNA modification is realized.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology technology, and in particular to a universal library construction method for detecting trace RNA modifications and its application in RNA modification detection. Background Art

[0002] There are more than 100 different types of post-transcriptional chemical modifications in RNA (e.g. 6 A、m 6 Am、m 5 C.hm 5 C、ac 4 C、m 1 A、m 7 G, Ψ, etc.), the four bases of RNA and ribose can all become targets for modification. People have come to realize that RNA not only acts as an intermediate and effector molecule in protein synthesis, but can also have a direct regulatory effect on gene expression.

[0003] There are some methods that have been reported in the field that can detect RNA chemical modifications at single-base resolution, quantification, and high-throughput across the entire transcriptome. Among them, single-base, high-throughput detection of RNA chemical modifications has greatly promoted research in related fields. However, there are still some areas for improvement in these methods, such as antibody-based enrichment of mRNA. 6 A-Rip-Seq technology (Nature 485, 201-206 (2012).; Cell 149, 1635-1646 (2012).) has a high false positive rate and a resolution of only 200nt. 6 A-SAC-seq technology 6 The A labeling process is relatively complicated (Nat Biotechnol. 2022 Aug; 40(8): 1210-1219.). GLORI chemical method for detecting m 6 A requires a sample starting amount of up to 200 ng, which severely limits the large-scale application of this method in biological systems (Nat Biotechnol. 2023 Mar; 41(3): 355-366.).

[0004] Therefore, based on the currently reported methods for detecting RNA modifications, it is more necessary to simplify the RNA library construction method by reducing the starting sample amount. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problems of high sample starting amount requirements and complex library construction in the prior art and to provide a universal library construction method for trace RNA modification detection and its application in RNA modification detection.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] One of the technical solutions of the present invention is to provide a method for constructing an RNA library enriched in trace amounts of RNA, comprising the following steps:

[0008] S1. Collect RNA samples to remove ribosomal RNA;

[0009] S2, performing end repair on the RNA sample obtained in step S1;

[0010] S3, using the RNA chain obtained in step S2 as a template, reverse transcribing into a first cDNA chain, and using the first cDNA chain as a template to synthesize a second cDNA chain complementary to the first cDNA chain, and hydrolyzing the template RNA chain;

[0011] S4, using the second cDNA obtained in step S3 as a template, in vitro transcription into an RNA chain;

[0012] S5, using the RNA chain obtained in step S4 as a template, reverse transcription is performed into a cDNA chain;

[0013] S6. Using the cDNA chain obtained in step S5 as a material for constructing an RNA library, amplifying the RNA library by PCR;

[0014] Wherein, step S2 is performed on the RNA sample obtained in step S1, or step S2 is performed after pre-treating the RNA epigenetic modification in the RNA sample obtained in step S1, or step S3 is performed after pre-treating the RNA epigenetic modification in the RNA chain obtained in step S2.

[0015] In some embodiments, in step S1, the RNA sample is selected from the group consisting of: an RNA sample from blood, an RNA sample from tissue, and an RNA sample from cells.

[0016] In some embodiments, the RNA epitope modifications in the RNA sample in step S1 or the RNA epitope modifications in the RNA chain obtained in step S2 are pretreated, and the pretreatment includes chemical treatment or enzymatic treatment of the RNA.

[0017] In some embodiments, the method for chemically treating RNA modifications comprises GLORI chemistry (recognition of m 6 A), BID-seq (identification of pseudouridine Ψ).

[0018] In some embodiments, in step S2, the RNA sample obtained in step S1 is subjected to end repair, specifically by dephosphorylating and polyadenylating the 3' ends of the fragmented RNA chains.

[0019] In some embodiments, the dephosphorylating nuclease is T4 polynucleotide kinase, and the polyadenylation nuclease is E. coli Poly(A) polymerase.

[0020] In some specific embodiments, in step S3, the RNA strand obtained in step S2 is used as a template for reverse transcription into a first cDNA strand, and the first cDNA strand is used as a template for synthesizing a second cDNA strand complementary to the first cDNA strand as follows:

[0021] S3-1 Template-displacement reverse transcription combined with second-strand cDNA synthesis

[0022] S3-1a. Using the RNA strand obtained in step S2 as a template and a T7 primer strand as a primer, a TSO strand is added to the reaction system. The T7 primer strand includes a T7 promoter, a cDNA adapter, and a polythymine strand whose end is complementary to the polyadenylate base at the 3' end of the RNA strand. The TSO strand is ligated to the 5' end of the RNA strand, thereby reversely transcribing to obtain a first cDNA strand (the 3' end of the synthesized first cDNA strand contains a complementary strand to the TSO strand), and the RNA strand is hydrolyzed.

[0023] S3-1b, using the first cDNA strand obtained in step S3-1a as a template and the TSO strand as a primer to extend and synthesize the second cDNA strand;

[0024] or,

[0025] S3-2 RNase H-dependent displacement synthesis of double-stranded cDNA

[0026] S3-2a, using the RNA strand obtained in step S2 as a template and a T7 primer strand as a primer, wherein the T7 primer strand includes a T7 promoter, a cDNA linker, and a polythymine at its end that complements the polyadenylate base at the 3' end of the RNA strand, thereby reversely transcribing to obtain a first cDNA strand, thereby obtaining an RNA-DNA hybrid strand;

[0027] S3-2b. Using the first cDNA strand obtained in step S3-2a as a template, RNase H is used to nick the RNA in the RNA-DNA hybrid strand. DNA polymerase I is then used to synthesize the second cDNA strand by replacing the RNA strand via a nick translation reaction.

[0028] In some specific embodiments, the nucleotide sequence of the T7 primer strand is shown as SEQ ID NO.1, and the nucleotide sequence of the TSO strand is shown as SEQ ID NO.2.

[0029] In some embodiments, in step S3-2b, the nucleases used to synthesize the blunt-ended second cDNA chain further include E.coil DNA ligase and T4 DNA polymerase.

[0030] In some embodiments, in step S3, the nuclease that hydrolyzes the template RNA chain is RNase H.

[0031] In some specific embodiments, in step S4, after the second cDNA obtained by methods S3-2a and S3-2b is used as a template and transcribed into an RNA chain in vitro, a 3' terminal adapter is connected to the end of the RNA chain to obtain an RNA chain with a 3' terminal adapter, and the nucleotide sequence of the 3' terminal adapter is shown in SEQ ID NO.3.

[0032] SEQ ID NO.3: / 5rApp / AGATCGGAAGAGCGTCGTG / 3Biotin /

[0033] In some embodiments, in step S4, the transcriptase used for in vitro transcription is T7 RNA polymerase.

[0034] In some specific embodiments, the ratio of the amount of RNA sample initially depleted of ribosomal RNA in step S1 to the amount of RNA obtained by in vitro transcription in step S4 is (0.1-10) ng: (5-500) ng.

[0035] In some specific embodiments, in step S5, the method of reverse transcribing the RNA chain obtained in step S4 as a template into a cDNA chain further includes: using RT Primer as a reverse transcription primer, and the nucleotide sequence of the RT Primer is shown in SEQ ID NO.4.

[0036] A second technical solution of the present invention is to provide an application of an RNA library prepared by the universal library construction method described in one of the above technical solutions in RNA modification detection.

[0037] In some embodiments, RNA modifications in the RNA library are detected using an Illumina or BGI sequencing platform.

[0038] The above-mentioned method for constructing an RNA library for detecting trace RNA modifications is named as the universal library construction method for ultra-trace epitranscriptome detection ( U universal u l tra-low i nput RNA library preparation method compatible with epiTranscriptome determination c tion strategies, Uli-epic), which involves treating RNA fragments with enzymes or chemicals required for RNA modification detection, then performing terminal dephosphorylation and Poly(A) tailing on the fragmented RNA. DNA primers containing a T7 promoter are then used for reverse transcription and double-stranded cDNA synthesis, ultimately forming double-stranded cDNA with a T7 promoter. The double-stranded cDNA is then subjected to in vitro transcription, RNA reverse transcription, and PCR library construction, followed by high-throughput sequencing and analysis of RNA modification data.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) The present invention provides a universal library construction method of Uli-epic, which adopts the core strategy of "low starting amount sample-chemical or enzymatic reaction-template replacement reverse transcription / RNase H-dependent replacement synthesis of double-stranded cDNA-T7 in vitro amplification-reverse transcription-PCR in vitro amplification". It can be used for high-throughput detection of RNA modification in low starting amount samples such as primary stem cells and eggs, and for detecting mRNA modifications in transcriptomes at the single base level. 6 A、m 1 A、m 7 The position and content of modifications such as G and Ψ can be determined, which enables the use of low-input RNA samples and quantitative detection of RNA modifications in the entire transcriptome at single-base resolution, further providing a high-throughput sequencing library construction method for in-depth exploration of low-sample-amount cells at the epigenetic regulation level.

[0041] (2) The present invention successfully reduced the amount of RNA used in this method to 100 pg by combining the Uli-epic universal library construction method with the RNA-seq method, and successfully performed high-throughput quantitative detection of gene expression of 100 pg HEK 293T mRNA at the whole transcriptome level.

[0042] (3) The present invention combines the Uli-epic universal library construction method with the GLORI method (Nat Biotechnol. 2023 Mar; 41(3): 355-366.) to successfully reduce the starting amount of RNA from 200 ng RNA to 10 ng, breaking through the bottleneck of RNA dosage and advancing it to the ultra-trace RNA single base m 6 A modification detection.

[0043] (4) The present invention successfully reduced the starting RNA amount from 10 ng RNA to 100 pg by combining the Uli-epic universal library construction method with BID-seq (Nat Biotechnol. 2023 Mar; 41(3): 344-354.), which advanced it to ultra-trace RNA single-base Ψ detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The left side shows the method for constructing an RNA library of trace RNA synthesized by template displacement reverse transcription and second-strand cDNA, and the right side shows the method for constructing an RNA library of trace RNA synthesized by RNase H-dependent displacement double-stranded cDNA.

[0045] Figure 2 This is the case of detecting a trace amount of HEK 293T mRNA transcriptome, i.e., Uli-epic RNA-seq, in Example 1. Double-stranded cDNA was synthesized by template displacement reverse transcription combined with second-strand cDNA synthesis.

[0046] Figure 3 Detection of trace Hela mRNA transcriptome m in Example 2 6 In the case of modification A, double-stranded cDNA was synthesized by template-displacement reverse transcription combined with second-strand cDNA synthesis.

[0047] Figure 4 In order to detect the Ψ modification of trace 293T mRNA transcriptome in Example 3, double-stranded cDNA was synthesized by template displacement reverse transcription combined with second-strand cDNA synthesis. DETAILED DESCRIPTION

[0048] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0049] In the following examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0050] Example 1:

[0051] This embodiment is based on Figure 1 The process shown on the left combines the Uli-epic library construction method with RNA-seq technology (referred to as Uli-RNA seq technology) to successfully perform high-throughput quantitative detection of gene expression of 100pg 293T mRNA at the whole transcriptome level ( Figure 2In this embodiment, the double-stranded cDNA synthesis method is a method of combining template replacement reverse transcription with second-strand cDNA synthesis.

[0052] The specific steps are as follows:

[0053] (1) Collection of RNA:

[0054] About 1000 adherent HeLa cells were cultured with PureLink TM Approximately 10 ng of total RNA was extracted using an RNA microextraction kit (ThermoFisher). Ribosomal RNA was removed using the Ribo-MagOff rRNA Depletion Kit (Vazyme) to yield approximately 100 pg of RNA. This 100 pg of RNA was diluted in 33.84 μL of RNase-free water, and 3.76 μL of 1 M NaHCO₃, pH 9.2, was added and reacted at 95°C for 5 minutes. Finally, 1 μL of 3 M NaOAc, pH 5.2, was added to neutralize the reaction.

[0055] (2) Perform 3'-end dephosphorylation of the RNA in step (1): T4 polynucleotide kinase is used as the nuclease. The reaction system configuration is shown in Table 1-1. The reaction system configured according to Table 1-1 is incubated at 37°C for 30 minutes and terminated at 65°C for 5 minutes to expose the 3'-terminal hydroxyl group (-OH) of the RNA.

[0056] Table 1-1

[0057]

[0058]

[0059] (3) Polyadenylation of the RNA obtained in step (2) at the 3' end: E. coli Poly(A) polymerase was used as the nuclease, and the reaction system configuration is shown in Table 1-2. The reaction system configured according to Table 1-2 was reacted at 37°C for 5 min, and then EDTA was added to a final concentration of 10 mM to terminate the reaction. Finally, the RNA was purified using Oligo Clean & Concentrator (Zymo Research).

[0060] Table 1-2

[0061] Components Volume (μL) Final concentration RNA obtained in step (2) 15 10×E.coli Poly(A)Polymerase Reaction Buffer 2 1× ATP (10 mM) 1.5 0.75mM E.coli Poly(A)Polymerase(5U / μL) 1 0.25 U / μL RNase inhibitor (40U / μL) 0.5 1U / μL Total volume 20

[0062] (4) Template-displacement reverse transcription and second-strand cDNA synthesis: Using the RNA obtained in step (3) as a template, a DNA primer with a T7 promoter and a polythymidine (oligo d(T)) at the 3' end of the primer is denatured and annealed with the polyadenosine (poly(A)) at the 3' end of the RNA obtained in step (3). The reaction system configuration is shown in Table 1-3. The reaction system configured according to Table 1-3 is reacted at 70°C for 5 minutes and then immediately placed on ice for 2-3 minutes.

[0063] Table 1-3

[0064] Components Volume (μL) Final concentration RNA obtained in step (3) 4 Primer (10 μM) 1 1 μM dNTP (10mM) 1 1mM Total volume 6

[0065] The primer (T7 primer chain) includes a T7 promoter, a cDNA linker, and polythymidine.

[0066] The nucleotide sequence is shown in SEQ ID NO.1:

[0067] 5'-GCCGCGAAATTAATACGACTCACTATAGGGATAATGAGCAGACGTGTGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTVN-3'.

[0068] The reaction system in Table 1-4 was added to the system after the reaction in Table 1-3, mixed, reacted at 42°C for 90 minutes, then at 85°C for 5 minutes, and finally quickly placed on ice for 2-3 minutes.

[0069] Table 1-4

[0070]

[0071]

[0072] The nucleotide sequence of the TSO chain is shown in SEQ ID NO.2:

[0073] 5'-GCTAATCATTGCACACGACGCTCTTCCGATCTrGrGrG-3', with biotin added to its 5' end.

[0074] During the above reaction, the reverse transcriptase adds several non-template nucleotides (primarily deoxycytidine C) to the transcribed strand after reaching the 5' end of the RNA template. These non-template nucleotides anneal with the TSO sequence (3' end of the TSO sequence is composed of three riboguanosines, rG), prompting the reverse transcriptase to switch from the RNA template to the TSO sequence. The 3' end of the first cDNA strand produced by reverse transcription contains the complement of the TSO sequence (CCC at the 3' end of the cDNA strand).

[0075] After reverse transcription is complete, RNase H is added to hydrolyze the RNA template. Simultaneously, DNA polymerase is used to extend the TSO strand to synthesize the second cDNA strand. The reaction system configuration is shown in Table 1-5. The reaction system configured in Table 1-5 is incubated at 37°C for 15 minutes to hydrolyze the RNA template. The reaction is then denatured at 95°C for 1 minute and then extended at 65°C for 10 minutes before termination.

[0076] Table 1-5

[0077] Components Volume (μL) Final concentration First strand cDNA 10 - Q5 Hot Start High Fidelity 2×Master Mix(NEB#M0494) 50 1× E. coli RNase H (5 U / μL) 5 0.25 U / μL <![CDATA[H2O]]> 35 - Total volume 100

[0078] After the reaction was complete, 2 μL of Exonuclease I (NEB, M0293) was added and the reaction was incubated at 37°C for 1 h to remove excess primers. Finally, the double-stranded cDNA was purified using Oligo Clean & Concentrator (Zymo Research).

[0079] (5) T7 in vitro transcription: Using the second cDNA obtained in step (4) as a template, RNA was transcribed in an in vitro cell-free system at 37°C to increase the amount of RNA. The T7 High Yield RNA Transcription Kit (Vazyme) was used. The reaction system configuration is shown in Table 1-6. The reaction system configured according to Table 1-6 was incubated at 37°C for 12 h.

[0080] Table 1-6

[0081]

[0082]

[0083] Add 2 μL DNase I to the reaction system and react at 37°C for 0.5 h to digest the double-stranded cDNA. TM MyOne TM RNA was purified using Silane (Thermo Fisher Scientific) to obtain 300-500 ng of RNA.

[0084] (6) Reverse transcription: Using the RNA obtained in step (5) as a template and RT Primer as a reverse transcription primer, reverse transcription is performed under the action of reverse transcriptase to obtain cDNA. The reaction system configuration is shown in Tables 1-7 and 1-8. First, the reaction system configured in Table 1-7 is reacted at 70°C for 5 minutes, then quickly placed on ice for 2-3 minutes, and then the reaction system configured in Table 1-8 is reacted at 50°C for 45 minutes, and then at 85°C for 2 minutes.

[0085] Table 1-7

[0086] Components Volume (μL) Final concentration RT Primer (50 μM) 1 3.85μM RNA obtained in step (5) 12 Total volume 13

[0087] Table 1-8

[0088] Components Volume (μL) Final concentration Table 1-7 Reaction solution after reaction 13 5×HiScript II Buffer 4 1× dNTP mix (10mM) 1 0.5mM HiScript II Reverse Transcriptase(200U / μL) 1 10 U / μL RNase inhibitor (40U / μL) 1 2U / μL Total volume 20

[0089] The nucleotide sequence of RT Primer is shown in SEQ ID NO.4:

[0090] ACACGACGCTCTTCCGATCT.

[0091] Add 1 μL RNase H to the reaction solution after the reaction in Table 1-8, incubate at 37°C for 0.5 h to digest RNA, and then use Dynabeads TM MyOne TM Single-stranded cDNA was purified using Silane (Thermo Fisher).

[0092] (7) Library construction: using NEB The single-stranded cDNA obtained in step (6) was amplified by PCR using Multiplex Oligos for Illumina to construct a library. The PCR product was purified using 1×AMPure beads (Thermo Fisher). The PCR product was electrophoresed on 1.5% agarose gel. After the electrophoresis was completed, the gel was cut and recovered, and the concentration was measured for high-throughput sequencing.

[0093] (8) After high-throughput sequencing, the data are analyzed using bioinformatics techniques.

[0094] like Figure 2 As shown:

[0095] Figure A is a scatter plot of gene expression levels detected by two biological replicates of bulk RNA-seq (RNA content of 10 ng). It can be seen from the figure that the reproducibility of bulk RNA-seq is very good.

[0096] Figure B is a scatter plot of gene expression levels detected by two biological replicates of Uli-RNA-seq (RNA content of 100 pg) in Example 1. It can be seen from the figure that Uli-RNA-seq has good repeatability.

[0097] Figure C is a scatter plot of gene expression levels of bulk RNA-seq and Uli-RNA-seq. It can be seen from the figure that the correlation between the expression of these two groups of genes is high.

[0098] Figure D shows the read coverage of all exons of the WDR75 gene in HEK 293T cells using two bulk RNA-seq and two Uli RNA-seq methods, indicating a high correlation between the gene expression levels identified by bulk RNA-seq and Uli RNA-seq.

[0099] Figure E shows the read coverage of all exons of the SERBP1 gene in HEK 293T cells using two bulk RNA-seq and two Uli RNA-seq methods, indicating a high correlation between the gene expression levels identified by bulk RNA-seq and Uli RNA-seq.

[0100] In summary, by combining the Uli-epic library construction method provided by the present invention with RNA-seq technology, gene expression level sequencing with ultra-low sample amounts can be achieved.

[0101] Example 2:

[0102] This embodiment is based on Figure 1 The process shown on the left combines the Uli-epic library construction method with the GLORI technology (referred to as Uli-GLORI technology) to successfully analyze the mRNA of 10ng Hela mRNA at the whole transcriptome level. 6 A modification for high-throughput single-base resolution quantitative detection ( Figure 3 In this embodiment, the double-stranded cDNA synthesis method is a method of combining template replacement reverse transcription with second-strand cDNA synthesis.

[0103] During the GLORI chemical reaction, unmethylated adenosine is efficiently deaminated to form inosine (A-to-I, >98%), which is read as guanosine (G) during sequencing, forming an A-to-G conversion; and m 6 A is still read as A after sequencing, thus achieving m 6 Single-base recognition of A. When the starting amount of RNA is extremely low, the GLORI chemistry will fragment the RNA, eliminating the need for a separate fragmentation step.

[0104] The specific steps are as follows:

[0105] (1) Collection of RNA:

[0106] About 10,000 adherent HeLa cells were cultured with PureLink TM About 100 ng of total RNA was extracted using an RNA microextraction kit (ThermoFisher), and ribosomal RNA was removed using a Ribo-MagOff rRNA Depletion Kit (Vazyme) to obtain about 10 ng of RNA.

[0107] (2) Preprocessing:

[0108] Add 0.1 ng of m-containing 6 The internal reference RNA (spike in RNA) at site A and the configuration of the reaction system are shown in Table 2-1. The reaction system configured according to Table 2-1 was reacted at 50°C for 30 minutes.

[0109] Table 2-1

[0110] Components Volume (μL) Final concentration RNA obtained in step (1) 14 10ng <![CDATA[Glyoxal solution(8.8M in H2O)]]> 6 1.32M DMSO 20 50% (v / v) Total volume 40

[0111] Add 10 μL of saturated H3BO3 solution to the reaction system in Table 2-1, react at 50°C for 30 minutes, then put on ice for 2-3 minutes, and finally mix with the reaction system shown in Table 2-2, react at 16°C for 8 hours, and purify RNA using Oligo Clean & Concentrator (Zymo Research).

[0112] Table 2-2

[0113] Components Volume (μL) Final concentration <![CDATA[5M NaNO2]]> 15 750mM 500mM MES buffer (pH 6.0) 8 40mM <![CDATA[Glyoxal solution(8.8M in H2O)]]> 10 0.88M <![CDATA[Nuclease free H2O]]> 17 Total volume 50

[0114] The purified RNA was treated with RNA Deprotection buffer at 95°C for 10 minutes, and then purified using Oligo Clean & Concentrator (Zymo Research).

[0115] The configuration of RNA Deprotection buffer is shown in Table 2-3:

[0116] Table 2-3

[0117] Components Volume (mL) Final concentration 1 M triethylammonium acetate solution pH 8.6 10 0.5M Deionized formamide 9.5 47.5% (v / v) <![CDATA[Nuclease free H2O]]> 0.5 Total volume 20

[0118] (3) The RNA obtained in step (2) was subjected to a 3'-end dephosphorylation reaction: the nuclease was T4 polynucleotide kinase, and the reaction system configuration was shown in Table 2-4. The reaction system configured according to Table 2-4 was incubated at 37°C for 30 minutes and terminated at 65°C for 5 minutes to expose the 3'-terminal hydroxyl group (-OH) of the RNA.

[0119] Table 2-4

[0120] Components Volume (μL) Final concentration RNA obtained in step (2) 12 μL 10×T4 Polynucleotide Kinase Reaction Buffer 1.5 μL 1× T4 Polynucleotide Kinase(10U / μL) 1 μL 0.66 U / μL SUPERase·In RNase Inhibitor(20U / μL) 0.5μL 0.66 U / μL Total volume 15 μL

[0121] (4) Polyadenylation of the RNA obtained in step (3) at the 3' end: E. coli Poly(A) polymerase was used as the nuclease, and the reaction system configuration is shown in Table 2-5. The reaction system configured according to Table 2-5 was reacted at 37°C for 5 min, and then EDTA was added to a final concentration of 10 mM to terminate the reaction. Finally, the RNA was purified using Oligo Clean & Concentrator (Zymo Research).

[0122] Table 2-5

[0123] Components Volume (μL) Final concentration RNA obtained in step (3) 15 10×E.coli Poly(A)Polymerase Reaction Buffer 2 1× ATP (10 mM) 1.5 0.75mM E.coli Poly(A)Polymerase(5U / μL) 1 0.25 U / μL RNase inhibitor (40U / μL) 0.5 1U / μL Total volume 20

[0124] (5) Template-displacement reverse transcription and second-strand cDNA synthesis: Using the RNA obtained in step (4) as a template, a DNA strand with a T7 promoter as a primer, with a polythymidine (oligo d(T)) at its 3' end, and the polyadenosine (poly(A)) at the 3' end of the RNA obtained in step (4) undergo denaturation annealing reaction. The reaction system configuration is shown in Table 2-6. The reaction system configured according to Table 2-6 is reacted at 70°C for 5 minutes and then immediately placed on ice for 2-3 minutes.

[0125] Table 2-6

[0126] Components Volume (μL) Final concentration RNA obtained in step (4) 4 Primer (10 μM) 1 1 μM dNTP (10mM) 1 1mM Total volume 6

[0127] The primer (T7 primer chain) includes a T7 promoter, a cDNA linker, and polythymidine.

[0128] The nucleotide sequence is shown in SEQ ID NO.1:

[0129] 5'-GCCGCGAAATTAATACGACTCACTATAGGGATAATGAGCAGACGTGTGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTVN-3'.

[0130] Add the reaction system in Table 2-7 to the system after the reaction in Table 2-6, mix, react at 42°C for 90 minutes, then react at 85°C for 5 minutes, and finally quickly place on ice for 2-3 minutes.

[0131] Table 2-7

[0132] Components Volume (μL) Final concentration Template Switching RT Buffer(4×) 2.5 1× Template Switching Oligo(TSO)(10μM) 0.5 0.5μM Template Switching RT Enzyme Mix(10×) 1 1× Total volume 4

[0133] The nucleotide sequence of the TSO chain is shown in SEQ ID NO.2:

[0134] 5'-GCTAATCATTGCACACGACGCTCTTCCGATCTrGrGrG-3', with biotin added to its 5' end.

[0135] During the above reaction, the reverse transcriptase adds several non-template nucleotides (primarily deoxycytidine) to the transcribed strand after reaching the 5' end of the RNA template. These non-template nucleotides anneal with the TSO sequence (3' end of the TSO sequence is composed of three riboguanosines, rG), prompting the reverse transcriptase to switch from the RNA template to the TSO sequence. The 3' end of the first cDNA strand produced by reverse transcription contains the complement of the TSO sequence (CCC at the 3' end of the cDNA strand).

[0136] After reverse transcription is complete, RNase H is added to hydrolyze the RNA template. Simultaneously, DNA polymerase is used to extend the TSO strand to synthesize the second cDNA strand. The reaction system configuration is shown in Table 2-8. The reaction system configured in Table 2-8 is incubated at 37°C for 15 minutes to hydrolyze the RNA template. The reaction is then denatured at 95°C for 1 minute and then extended at 65°C for 10 minutes before termination.

[0137] Table 2-8

[0138]

[0139]

[0140] After the reaction was complete, 2 μL of Exonuclease I (NEB, M0293) was added and the reaction was incubated at 37°C for 1 h to remove excess primers. Finally, the double-stranded cDNA was purified using Oligo Clean & Concentrator (Zymo Research).

[0141] (6) T7 in vitro transcription: Using the second cDNA obtained in step (5) as a template, RNA was transcribed in an in vitro cell-free system at 37°C to increase the amount of RNA. The T7 High Yield RNA Transcription Kit (Vazyme) was used. The reaction system configuration is shown in Table 2-9. The reaction system configured according to Table 2-9 was incubated at 37°C for 12 h.

[0142] Table 2-9

[0143] Components Volume (μL) Final concentration 10×Transcription buffer 2 1× ATP Solution (100 mM) 2 10 mM GTP Solution (100 mM) 2 10 mM CTP Solution (100 mM) 2 10 mM TTP Solution (100 mM) 2 10 mM Double-stranded cDNA from step (5) 7 RNase inhibitor (40 U / μL) 0.8 1.6 U / μL Inorganic Pyrophosphatase 0.1 U / μL 0.2 T7 RNA Polymerase 2 Rnase free water 0 Total volume 20

[0144] Add 2 μL DNase I to the reaction system and react at 37°C for 0.5 h to digest the double-stranded cDNA. TM MyOne TM RNA was purified using Silane (Thermo Fisher Scientific) to obtain 300-500 ng of RNA.

[0145] (7) Reverse transcription: Using the RNA obtained in step (6) as a template and RT Primer as a reverse transcription primer, reverse transcription is performed under the action of reverse transcriptase to obtain cDNA. The reaction system configuration is shown in Tables 2-10 and 2-11. First, the reaction system configured in Table 1-10 is reacted at 70°C for 5 minutes, then quickly placed on ice for 2-3 minutes. Then, the reaction system configured in Table 2-11 is reacted at 50°C for 45 minutes, and then at 85°C for 2 minutes.

[0146] Table 2-10

[0147] Components Volume (μL) Final concentration RT Primer (50 μM) 1 3.85μM RNA obtained in step (7) 12 Total volume 13

[0148] Table 2-11

[0149]

[0150]

[0151] The nucleotide sequence of RT Primer is shown in SEQ ID NO.4:

[0152] ACACGACGCTCTTCCGATCT.

[0153] Add 1 μL RNase H to the reaction solution after the reaction in Table 2-11, incubate at 37°C for 0.5 h to digest RNA, and then use Dynabeads TM MyOne TM Single-stranded cDNA was purified using Silane (Thermo Fisher).

[0154] (8) Library construction: using NEB The single-stranded cDNA obtained in step (7) was amplified by PCR using Multiplex Oligos for Illumina to construct a library. The PCR product was purified using 1×AMPure beads (Thermo Fisher). The PCR product was electrophoresed on 1.5% agarose gel. After the gel was cut and recovered after electrophoresis, the concentration was measured and high-throughput sequencing was performed.

[0155] (9) After high-throughput sequencing, the data are analyzed using bioinformatics techniques.

[0156] like Figure 3 As shown:

[0157] Figure A is a quality control chart after library construction. It can be seen from the figure that the library quality is good, and the fragment size distribution is between 200-500bp.

[0158] Figure B shows the bioinformatics analysis of trace Hela mRNA m 6 The region where point A is enriched is near the CDS termination region.

[0159] Figure C shows the m 6 Analysis of the A site motif revealed m 6 The most common motif in the A site is GGm 6 ACU.

[0160] Figure D shows the identification of m 6 The upstream and downstream base sequences of the A site indicate that GGm 6 ACU、GAm 6 ACU、AGm 6 ACU, GGm 6 ACA、TGm 6 ACU is the most common of the five m 6 A-site motif.

[0161] Figure E shows the sequence information of the internal reference RNA (spike in RNA) (containing an m 6 A base), after high-throughput sequencing, the data displayed by IGV, as can be seen from the figure, the m in the original sequence 6 A single base is displayed as A, while A in the original sequence is displayed as G.

[0162] Figure F shows the sequence information of MRPS26 gene (containing multiple m 6 A base), after high-throughput sequencing, the data displayed by IGV shows that there are three m 6 A site.

[0163] In summary, by combining the Uli-epic library construction method provided by the present invention with the GLORI technology, high-throughput detection with reduced starting sample size is achieved without affecting the GLORI technology's ability to detect RNA modifications at single bases, thus solving the problem of the GLORI technology requiring a large starting RNA sample size.

[0164] Example 3:

[0165] This embodiment is based on Figure 1 As shown in the process on the left, the present invention successfully reduced the RNA usage of this method from 10 ng RNA to 100 pg by combining the Uli-epic universal library construction method with BID-seq (Nat Biotechnology. 2023 Mar; 41(3): 344-354.), pushing it to the ultra-trace RNA single-base Ψ detection level.

[0166] The specific steps are as follows:

[0167] (1) Collection of RNA:

[0168] About 1000 adherent 293T cells were cultured with PureLink TM Approximately 10 ng of total RNA was extracted using an RNA microextraction kit (ThermoFisher). Ribosomal RNA was removed using the Ribo-MagOff rRNA Depletion Kit (Vazyme), yielding approximately 100 pg and 1 ng of RNA, respectively. The RNA was diluted in 33.84 μL of RNase-free water, and 3.76 μL of 1 M NaHCO₃, pH 9.2, was added and reacted at 95°C for 5 minutes. Finally, 1 μL of 3 M NaOAc, pH 5.2, was added to neutralize the reaction.

[0169] (2) Perform 3'-end dephosphorylation of the RNA in step (1): T4 polynucleotide kinase is used as the nuclease. The reaction system configuration is shown in Table 3-1. The reaction system configured according to Table 3-1 is incubated at 37°C for 30 minutes and terminated at 65°C for 5 minutes to expose the 3'-terminal hydroxyl group (-OH) of the RNA.

[0170] Table 3-1

[0171]

[0172] (3) The RNA obtained in step (2) was subjected to 3 ’Terminal polyadenylation: The nuclease used was E. coli Poly(A) polymerase. The reaction system configuration is shown in Table 3-2. The reaction system configured according to Table 3-2 was incubated at 37°C for 5 min. EDTA was then added to a final concentration of 10 mM to terminate the reaction. RNA was purified using Oligo Clean & Concentrator (Zymo Research).

[0173] Table 3-2

[0174] Components Volume (μL) Final concentration RNA obtained in step (2) 15 10×E.coli Poly(A)Polymerase Reaction Buffer 2 1× ATP (10 mM) 1.5 0.75mM E.coli Poly(A)Polymerase(5U / μL) 1 0.25 U / μL RNase inhibitor (40U / μL) 0.5 1U / μL Total volume 20

[0175] (4) The RNA purified from step (3) was subjected to a sodium bisulfite-Ψ addition reaction. 270 mg of Na2SO3 and 34 mg of NaHSO3 were dissolved in 900 μL of RNase-free water to prepare a BS solution. 5 μL of RNA was mixed with 45 μL of BS solution and reacted at 70°C for 3 h. Desulfurization was performed using the EZ RNA Methylation kit (Zymo Research, #R5001), and the RNA was purified.

[0176] (5) Template-displacement reverse transcription and second-strand cDNA synthesis: Using the RNA obtained in step (4) as a template, a DNA strand with a T7 promoter as a primer, with a polythymidine (oligo d(T)) at its 3' end, and the polyadenosine (poly(A)) at the 3' end of the RNA obtained in step (4) undergo denaturation annealing reaction. The reaction system configuration is shown in Table 3-3. The reaction system configured according to Table 3-3 is reacted at 70°C for 5 minutes and then immediately placed on ice for 2-3 minutes.

[0177] Table 3-3

[0178]

[0179]

[0180] The primer (T7 primer chain, T7 primer) includes T7 promoter, cDNA linker and polythymidine connected in sequence.

[0181] The nucleotide sequence is shown in SEQ ID NO.1:

[0182] 5'-GCCGCGAAATTAATACGACTCACTATAGGGATAATGAGCAGACGTGTGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTVN-3'.

[0183] Add the reaction system in Table 3-4 to the system after the reaction in Table 3-3, mix, react at 42°C for 90 minutes, then react at 85°C for 5 minutes, and finally quickly place on ice for 2-3 minutes.

[0184] Table 3-4

[0185] Components Volume (μL) Final concentration Template Switching RT Buffer(4×) 2.5 1× Template Switching Oligo(TSO)(10μM) 0.5 0.5μM Template Switching RT Enzyme Mix(10×) 1 1× Total volume 4

[0186] The nucleotide sequence of the TSO chain is shown in SEQ ID NO.2:

[0187] 5'-GCTAATCATTGCACACGACGCTCTTCCGATCTrGrGrG-3', with biotin added to its 5' end.

[0188] During the above reaction, the reverse transcriptase adds several non-template nucleotides (primarily deoxycytidine) to the transcribed strand after reaching the 5' end of the RNA template. These non-template nucleotides anneal with the TSO sequence (3' end of the TSO sequence is composed of three riboguanosines, rG), prompting the reverse transcriptase to switch from the RNA template to the TSO sequence. The 3' end of the first cDNA strand produced by reverse transcription contains the complement of the TSO sequence (CCC at the 3' end of the cDNA strand).

[0189] After reverse transcription is complete, RNase H is added to hydrolyze the RNA template. Simultaneously, DNA polymerase is used to extend the TSO strand to synthesize the second cDNA strand. The reaction system configuration is shown in Table 3-5. The reaction system configured in Table 3-5 is incubated at 37°C for 15 minutes to hydrolyze the RNA template. The reaction is then denatured at 95°C for 1 minute and then extended at 65°C for 10 minutes before termination.

[0190] Table 3-5

[0191] Components Volume (μL) Final concentration First strand cDNA 10 - Q5 Hot Start High Fidelity 2×Master Mix(NEB#M0494) 50 1× E. coli RNase H (5U / μL) 5 0.25 U / μL <![CDATA[H2O]]> 35 - Total volume 100

[0192] After the reaction was complete, 2 μL of Exonuclease I (NEB, M0293) was added and the reaction was incubated at 37°C for 1 hour to remove excess primers. Finally, the double-stranded cDNA was purified using Oligo Clean & Concentrator (Zymo Research).

[0193] (6) T7 in vitro transcription: Using the second cDNA obtained in step (5) as a template, RNA was transcribed in an in vitro cell-free system at 37°C to increase the total amount of RNA product. The T7 High Yield RNA Transcription Kit (Vazyme) was used. The reaction system configuration is shown in Table 3-6. The reaction system configured according to Table 3-6 was incubated at 37°C for 12 hours.

[0194] Table 3-6

[0195] Components Volume (μL) Final concentration 10×Transcription buffer 2 1× ATP Solution (100mM) 2 10mM GTP Solution (100mM) 2 10mM CTP Solution (100mM) 2 10mM TTP Solution (100mM) 2 10mM Double-stranded cDNA from step (5) 7 RNase inhibitor (40U / μL) 0.8 1.6 U / μL Inorganic Pyrophosphatase 0.1U / μL 0.2 T7 RNA Polymerase 2 RNase-free water 0 Total volume 20

[0196] Add 2 μL DNase I to the reaction system and react at 37°C for 0.5 h to digest the double-stranded cDNA. TM MyOne TM RNA was purified using Silane (Thermo Fisher Scientific) to obtain 300-500 ng of RNA.

[0197] (7) Reverse transcription: Using the RNA obtained in step (6) as a template and RT Primer as a reverse transcription primer, reverse transcription was performed under the action of reverse transcriptase to obtain cDNA. The reaction system configuration is shown in Tables 3-7 and 3-8. First, the reaction system configured according to Table 1-7 was reacted at 70°C for 5 minutes, then quickly placed on ice for 2-3 minutes, and then the reaction system configured according to Table 3-8 was reacted at 50°C for 45 minutes, and then at 85°C for 2 minutes.

[0198] Table 3-7

[0199] Components Volume (μL) Final concentration RT Primer (50 μM) 1 3.85μM RNA obtained in step (6) 12 Total volume 13

[0200] Table 3-8

[0201] Components Volume (μL) Final concentration Table 3-7 Reaction solution after reaction 13 5×HiScript II Buffer 4 1× dNTP mix (10mM) 1 0.5mM HiScript II Reverse Transcriptase(200U / μL) 1 10 U / μL RNase inhibitor (40U / μL) 1 2U / μL Total volume 20

[0202] The nucleotide sequence of RT Primer is shown in SEQ ID NO.4:

[0203] ACACGACGCTCTTCCGATCT.

[0204] Add 1 μL RNase H to the reaction solution after the reaction in Table 3-8, incubate at 37°C for 0.5 h to digest RNA, and then use Dynabeads TM MyOne TM Single-stranded cDNA was purified using Silane (Thermo Fisher).

[0205] (8) Library construction: using NEB The single-stranded cDNA obtained in step (7) was amplified by PCR using Multiplex Oligos for Illumina to construct a library. The PCR product was purified using 1×AMPure beads (Thermo Fisher). The PCR product was electrophoresed on 1.5% agarose gel. After the gel was cut and recovered after electrophoresis, the concentration was measured and high-throughput sequencing was performed.

[0206] (9) After high-throughput sequencing, the data are analyzed using bioinformatics techniques.

[0207] like Figure 4 As shown:

[0208] A Venn diagram showing the overlap of Ψ modifications detected by two biological replicates of bulk BID-seq at the single-base level.

[0209] B Venn diagram showing the overlap of Ψ modifications detected at the single base level by two biological replicates of Uli BID-seq using 1 ng of RNA.

[0210] C Venn diagram showing the overlap of Ψ modifications detected at the single base level by two biological replicates of Uli BID-seq on 100 pg RNA.

[0211] D Venn diagram showing the overlap of Ψ modifications detected at the single base level by bulk BID-seq, Uli BID-seq of 1 ng RNA, and Uli BID-seq of 100 pg RNA.

[0212] Figure E shows the sequence information of the internal reference RNA (spike in RNA) (containing a single Ψ base). After bulk BID-seq and Uli BID-seq high-throughput sequencing, the data is displayed via IGV. As can be seen from the figure, the single Ψ base in the original sequence is displayed as a deletion mutation, while the Ψ in the input control group is displayed as T.

[0213] Figure F shows the upstream and downstream base sequences of Ψ modification sites detected by bulk BID-seq, indicating that GCΨCT, GAΨGG, GAΨGC, AAΨGG, and GTΨGG are the five most common Ψ site motifs.

[0214] Figure G shows the upstream and downstream base sequences of the Ψ modification sites detected by Uli BID-seq on 1 ng of RNA. It shows that GTΨGG, GGΨGA, GCΨGC, TTΨGG, and GAΨCA are the five most common Ψ site motifs.

[0215] Figure H shows the upstream and downstream base sequences of the Ψ modification sites detected by Uli BID-seq on 100 pg RNA, indicating that TTΨGT, TGΨGA, CTΨAG, ACΨGA, and TTΨGG are the five most common Ψ site motifs.

[0216] The FG diagram shows that bulk BID-seq and Uli BID-seq have different preferences for identifying motifs at Ψ modification sites.

[0217] In summary, by combining the Uli-epic library construction method provided by the present invention with the BID-seq technology, high-throughput detection with reduced starting sample size is achieved without affecting the efficiency of the BID-seq technology in single-base detection of RNAΨ modifications, and the starting RNA sample amount in the BID-seq technology is pushed to ultra-trace amounts (1 ng-100 pg).

[0218] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A universal library construction method for detecting trace RNA modifications, characterized in that: The steps include: S1. Collect RNA samples to remove ribosomal RNA; S2, performing end repair on the RNA sample obtained in step S1; S3, using the RNA chain obtained in step S2 as a template, reverse transcribing into a first cDNA chain, and using the first cDNA chain as a template to synthesize a second cDNA chain complementary to the first cDNA chain, and hydrolyzing the template RNA chain; S4, using the second cDNA obtained in step S3 as a template, in vitro transcription into an RNA chain; S5, using the RNA chain obtained in step S4 as a template, reverse transcription is performed into a cDNA chain; S6. Using the cDNA chain obtained in step S5 as a material for constructing an RNA library, amplifying the RNA library by PCR; Wherein, step S2 is performed on the RNA sample obtained in step S1, or step S2 is performed after pre-treating the RNA epigenetic modification in the RNA sample obtained in step S1, or step S3 is performed after pre-treating the RNA epigenetic modification in the RNA chain obtained in step S2.

2. A universal library construction method for detecting trace RNA modifications according to claim 1, characterized in that: In step S1, the RNA sample is selected from the following group: an RNA sample from blood, an RNA sample from tissue, and an RNA sample from cells.

3. A universal library construction method for detecting trace RNA modifications according to claim 1, characterized in that: The RNA epigenetic modifications in the RNA sample in step S1 or the RNA epigenetic modifications in the RNA chain obtained in step S2 are pretreated, and the pretreatment includes chemical treatment or enzymatic treatment of the RNA.

4. A universal library construction method for detecting trace RNA modifications according to claim 3, characterized in that: Methods for chemical manipulation of RNA modifications include GLORI chemistry and BID-seq.

5. A universal library construction method for detecting trace RNA modifications according to claim 1, characterized in that: In step S2, the RNA sample obtained in step S1 is subjected to end repair, specifically, the 3' ends of the fragmented RNA chains are dephosphorylated and polyadenylated.

6. A universal library construction method for detecting trace RNA modifications according to claim 1, characterized in that: In step S3, the RNA strand obtained in step S2 is used as a template for reverse transcription into a first cDNA strand, and the first cDNA strand is used as a template for synthesizing a second cDNA strand complementary to the first cDNA strand. Specifically, the method is as follows: S3-1a. Using the RNA strand obtained in step S2 as a template and a T7 primer strand as a primer, a TSO strand is added to the reaction system. The T7 primer strand includes a T7 promoter, a cDNA adapter, and a polythymine strand whose end is complementary to the polyadenylate base at the 3' end of the RNA strand. The TSO strand is ligated to the 5' end of the RNA strand, thereby reverse transcribing to obtain a first cDNA strand, and hydrolyzing the RNA strand. S3-1b, using the first cDNA strand obtained in step S3-1a as a template and the TSO strand as a primer to extend and synthesize the second cDNA strand; or, S3-2a, using the RNA strand obtained in step S2 as a template and a T7 primer strand as a primer, wherein the T7 primer strand includes a T7 promoter, a cDNA linker, and a polythymine at its end that complements the polyadenylate base at the 3' end of the RNA strand, thereby reversely transcribing to obtain a first cDNA strand, thereby obtaining an RNA-DNA hybrid strand; S3-2b. Using the first cDNA chain obtained in step S3-2a as a template, RNase H is used to nick the RNA in the RNA-DNA hybrid chain, and then DNA polymerase I is used to replace the RNA chain by a nick-filling reaction to synthesize the second cDNA chain.

7. A universal library construction method for detecting trace RNA modifications according to claim 6, characterized in that: The nucleotide sequence of the T7 primer chain is shown in SEQ ID NO. 1, and the nucleotide sequence of the TSO chain is shown in SEQ ID NO.

2.

8. A universal library construction method for detecting trace RNA modifications according to claim 6, characterized in that: In step S4, the double-stranded cDNA obtained by methods S3-2a and S3-2b is used as a template to be transcribed into an RNA chain in vitro, and a 3' terminal adapter is connected to the end of the RNA chain to obtain an RNA chain with a 3' terminal adapter. The nucleotide sequence of the 3' terminal adapter is shown in SEQ ID NO.

3.

9. A universal library construction method for detecting trace RNA modifications according to claim 1, characterized in that: The ratio of the amount of RNA sample initially depleted of ribosomal RNA in step S1 to the amount of RNA obtained by in vitro transcription in step S4 is (0.1-10) ng: (5-500) ng.

10. Use of an RNA library prepared by the universal library construction method according to any one of claims 1 to 9 in RNA modification detection.

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