CN113584135A

CN113584135A - Method for mixed sample detection of RNA modification and realization of accurate quantification

Info

Publication number: CN113584135A
Application number: CN202110844695.1A
Authority: CN
Inventors: 骆观正; 陈涛; 谢应源; 张璋; 陈鸿萱; 刘标地
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2021-07-26
Filing date: 2021-07-26
Publication date: 2021-11-02
Anticipated expiration: 2041-07-26
Also published as: CN113584135B

Abstract

The invention belongs to the technical field of RNA modification site detection, and particularly relates to a method for detecting RNA modification and realizing accurate quantification by using a mixed sample. Because the mixed sample is used for immunoprecipitation, the total amount of the mixed sample only needs to reach the minimum standard of the input amount of the immunoprecipitation method, so that the input amount of a single sample can be greatly reduced, the MeRIP detection of a rare sample becomes possible, the detection cost and the experimental labor amount are reduced, errors caused by batch effect of experiments can be avoided, and the m6A content in the sample can be compared among groups. It can be seen that the present invention enables parallel detection and accurate quantification of m6A modifications on RNA in different samples under the same conditions.

Description

Method for mixed sample detection of RNA modification and realization of accurate quantification

Technical Field

The invention belongs to the technical field of RNA modification site detection, and particularly relates to a method for detecting RNA modification by mixing samples and realizing accurate quantification.

Background

Over 100 modifications are currently known to exist on RNA, including: n6-adenylate methylation (m6A), N1-adenylate methylation (m1A), cytosine hydroxylation (m5C) and the like. Among them, m6A (N6-methylaldenosine, N6-methyladenine) is a modification of adenine nucleotide under the catalysis of adenylate methyltransferase, is one of the most abundant RNA modified bases in eukaryotic mRNA, and is generally distributed in different types of RNA. In recent years, several studies have demonstrated that RNA methylation plays an important role in variable cleavage of RNA, transcription initiation and translation. The methylation level of RNA is closely related to the biological processes of metabolism, genetic development, disease occurrence and development and the like.

At present, most methods for identifying the m6A site rely on antibody recognition and immunoprecipitation technology, and the most widely used is Methylated RNA immunization and sequencing (MeRIP-seq or m6A-seq) technology. However, MeRIP-seq based on immunoprecipitation requires a large sample input (generally more than 500. mu.g of total RNA is required), and thus cannot be applied to some rare samples; moreover, because of certain differences in the efficiency of antibodies of different batches, the conditions adopted in each experiment cannot be completely consistent, so that the repeatability of the method is poor, and the comparison between samples has an error which is difficult to eliminate, thereby greatly limiting the application of the m6A modification detection technology in clinical and basic research. Therefore, it is necessary to provide a method for detecting m6A modification with an ultra-low initial amount.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a method for detecting RNA modification by mixing samples and realizing accurate quantification, which can carry out parallel detection on m6A modification on RNA in different samples under the same condition and realize accurate quantification, thereby greatly reducing the input amount of a single sample, the detection cost and the labor amount, effectively avoiding errors caused by different experimenters and antibody batch effects, and being applied to the detection aspect of m6A locus of some rare samples.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides a method for detecting RNA modification and realizing accurate quantification by mixing samples, which comprises the following steps:

s1 synthesizing a product containing a Barcode sequence and having NH at the 3' end₂C6 modified and 3 'end connector with phosphoric acid modified 5' end, and the 3 'end connector is processed with 5' end adenylation treatment to facilitate subsequent connection reaction;

s2, connecting the end connector containing the Barcode3 'after the adenylation with the 3' tail ends of different RNA samples, and labeling the RNA samples with the Barcode;

s3, mixing different RNA samples with Barcode together to perform an immunoprecipitation reaction;

s4, after immunoprecipitation reaction, connecting the 5 'end of the RNA fragment with a 5' adaptor, performing reverse transcription by using a universal primer, and finally performing library amplification sequencing and data analysis to obtain the modification condition of m 6A.

Preferably, the Barcode sequence is a 6 base Barcode or a 2 random base +6 base Barcode.

Preferably, the 3' end fitting includes RLA3(NEB) -Barcode1 shown in SEQ ID NO.1, RLA3(NEB) -Barcode2 shown in SEQ ID NO.2, RLA3(NEB) -Barcode3 shown in SEQ ID NO.3, and RLA3(NEB) -Barcode4 shown in SEQ ID NO. 4. The 3' terminal connector sequence is a preferred embodiment of the present invention, but is not limited to these sequences, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and shall be included in the scope of the present invention.

Preferably, step S1 is to perform 5 'terminal adenylation treatment on the 3' end linker using Mth RNA ligase.

The invention uses a 3' end connector with a special modification at the tail end, the phosphorylation of the 5 ' end of the connector can lead Mth RNA ligase to carry out the adenylation treatment, and the NH of the 3' end of the connector₂The C6 modification prevents the linker from self-ligating.

Preferably, fragmentation and end repair of the RNA sample is required before the end linker containing Barcode3 'after adenylation is ligated to the 3' ends of different RNA samples.

Preferably, step S2 is to ligate the adenylated Barcode3 'containing end linker to the 3' end of the RNA sample using KQ mutated T4RNA ligase.

Preferably, step S4 is to use the 5' end band with NH₂C6 modified 5 ' adaptor the 5 ' adaptor was ligated to the 5 ' end of the RNA fragment by T4RNA Ligase1, followed by reverse transcription using the universal primers and finally library amplification without purification.

Preferably, the sequence of the 5' linker is shown as SEQ ID NO.5, and the sequence of the universal primer is shown as SEQ ID NO. 6.

Preferably, in step S4, the data analysis includes splitting the mixed sample data by the Barcode sequence, comparing the RNA samples, identifying peak calling and m6A loci, and finally performing differential methylation analysis by combining the results of the samples.

Because the experimental process of the enrichment of the antibody of each sample is the same in the process of carrying out the immunoprecipitation reaction by adopting the method of the invention, the differential methylation analysis and the quantification among the samples can be carried out after the data of each sample is resolved by Barcode.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a method for detecting m6A modification by using Barcode labeling mixed samples, which comprises the steps of labeling a plurality of samples by using Barcode, mixing the samples, simultaneously carrying out MeRIP-seq, carrying out sequencing, and splitting each sample by using a Barcode sequence for analysis and comparison. Because the invention uses the mixed sample for immunoprecipitation, the total amount of the mixed sample only needs to reach the minimum standard (500 ng) of the input amount of the immunoprecipitation method, thereby greatly reducing the input amount of a single sample, enabling the MeRIP detection of rare samples to be possible, and simultaneously reducing the detection cost and the experimental labor amount. More importantly, the experiment is carried out on different samples simultaneously, so that the error caused by batch effect of the experiment can be avoided, and the m6A content in the samples can be compared among groups, thereby achieving the quantitative purpose. Therefore, the method can carry out parallel detection on the m6A modification on the RNA in different samples under the same condition and realize accurate quantification, thereby effectively avoiding errors caused by different experimenters and antibody batch effects. Compared with the original MeRIP-seq, the method greatly reduces the input amount of a single sample, the detection cost and the detection labor amount, can be applied to the detection of m6A locus of some rare samples, and can be popularized to other RNA modification detection methods depending on antibodies.

Drawings

FIG. 1 is a flow chart of the mixed sample detection m6A modification;

FIG. 2 is a plot of the m6A distribution for both the mES and 293T samples;

FIG. 3 is a motif analysis of both mES and 293T samples;

FIG. 4 shows the distribution pattern of m6A of four mES wild type, Wtap knockout, Mettl3 knockout and Mettl14 knockout samples;

FIG. 5 is an m6A motif analysis of four mES wild type, Wtap knockout, Mettl3 knockout and Mettl14 knockout samples;

FIG. 6 is a quantitative difference analysis of M6A for M3KO-WT versus WT.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.

Example 1A method for mixed sample detection of m6A modification by Barcode labeling

The experimental flow of the method is shown in fig. 1, and specifically comprises the following steps:

(1) oligo Barcode labeling and Synthesis of Universal primers

1) Synthesis of Oligo Barcode tags (i.e., Barcode-containing 3' end linkers)

Synthesizing a 3' end NH containing Barcode sequence₂The 3 'end connector which is modified by C6 and modified by 5' end with phosphoric acid is designed and synthesized by NEB company, and the specific sequence details and sequence characteristics are shown in the following table:

2) the synthesis of the universal primer, the details of the sequence and the sequence characteristics are shown in the following table:

(2) adenylation treatment of end connector containing barcode3

Each Oligo Barcode labeled 3 'end linker requires 5' end adenylation before subsequent ligation reactions can be performed, and the system of a single adenylation reaction is shown in the following table:

the reaction conditions for the single adenylation are shown in the following table:

65℃	1 hour
		85℃	5 minutes

After the reaction was completed, purification was performed using Oligo clean & concentrator-5(Zymo Research), and 15. mu.L of RNase-free water was eluted, aliquoted and stored in a refrigerator at-80 ℃.

(3) RNA fragmentation

Selecting two mRNA samples (both RNA samples with rRNA removed) of mES wild type mRNA (derived from mouse cells and cells purchased from a cell bank of Chinese academy of sciences) and 293T mRNA (293T derived from human embryonic kidney cells and cells purchased from a cell bank of Chinese academy of sciences), and respectively carrying out fragmentation treatment on each RNA sample, wherein the reaction system of single fragmentation is shown in the following table:

RNA sample (mES wild type/293T mRNA sample)	Within 1 mu g
		10×RNA fragmentation reagents(ThermoFisher)	1μL
RNase-free water	To 10μL

The reaction system was incubated at 94 ℃ for 30 seconds, immediately placed on ice after the reaction was completed, and 1. mu.L of Stop solution was added to terminate the fragmentation reaction. And purified using RNA clean & purifier-5 (Zymo Research), taking 15. mu.L of RNase-free water eluate.

(4) End repair of fragmentation products

And (3) repairing each RNA fragmentation product in the step (3), wherein the reaction system of single repair is shown as the following table:

the reaction conditions for a single repair are shown in the table below:

37℃	30 minutes
		65℃	10 minutes
4℃	-

After completion of the reaction, the reaction mixture was purified using RNA clean & concentrator-5(Zymo Research), and 9. mu.L of RNase-free water was eluted. And detecting the concentration of each sample, and adjusting the input amount of each sample to ensure that the input amount of each sample is relatively balanced, wherein the total amount of all samples is not less than 500 ng.

(5) Barcode labeling of different samples

And (3) respectively connecting each mRNA sample repaired in the step (4) with an adenylated Barcode3 'end connector, wherein the connection reaction system of the single 3' end connector (containing Barcode) is shown as the following table (namely, the Barcode1 is adopted to mark mES wild type mRNA, and the Barcode2 is adopted to mark 293T mRNA):

the reaction was incubated at 25 ℃ for 2 hours and then at 4 ℃ overnight.

After the reaction is finished, the excess 3' end joint is removed, and the reaction system except the excess joint is shown in the following table:

ligation product of the above step	30μL
		Lambda Exonuclease(NEB)	2μL
5`Deadenylase(NEB)	1μL

The reaction conditions except for the excess linker are shown in the following table:

30℃	30 minutes
		37℃	30 minutes
70℃	5 minutes

After the reaction is finished, all the Barcode-labeled RNA samples are mixed, purified by using RNA clean & concentrator-5(Zymo Research), 20 mu L of RNase-free water is taken to elute all the products, and the concentration is detected to ensure that the total amount of each Barcode-labeled RNA sample is more than 500ng (in the case of shortage, a proper amount of RNA Spike-in can be added).

(6) Mixed sample MeRIP

The buffer components used in this step are shown in the following table:

1) 500 ng-1. mu.g of the RNA mixture purified in step (5) and labeled with Barcode was measured as a substrate for immunoprecipitation, and the following operations were carried out (1/10 of the total amount of RNA substrate was taken as an Input sample, 9/10 was taken as an Ip sample):

25. mu.L of Protein G magnetic beads equilibrated at room temperature were taken, added with 250. mu.L of Reaction buffer and 2. mu. L m6A antibody (NEB), and incubated at 4 ℃ for 40-60 minutes on a rotator.

② remove the supernatant, using 250 u L Reaction buffer washing magnetic beads twice, finally using 250 u L Reaction buffer heavy suspension magnetic beads, adding IP samples, at 4 degrees C under the condition of 2 hours incubation on the rotating instrument.

③ removing the supernatant, and washing with 250. mu.L of Reaction buffer, Low-salt buffer and High-salt buffer in sequence, wherein each buffer is washed twice, 5 minutes each time.

(iv) eluting the IP product with 50. mu.L Buffer RLT (QIAGEN, Cat. No./ID:79216), this step was performed twice, the two products were combined and purified with RNA clean & controller-5 (Zymo Research), and 8. mu.L of RNase-free water was used to elute the IP product.

2) Connection 5' end joint

The 5' end adapter (RLA5) was incubated at 70 ℃ for 3 minutes, immediately on ice. Then, the IP product obtained by the above purification or the initially retained Input sample is used as a template to perform a 5' end linker ligation reaction, and the reaction systems are shown in the following table:

purified IP product/initial retained Input sample	7μL
			10×T4 RNA Ligation buffer(NEB)	2μL
50％PEG 8000	8μL
		5' end connector (RLA5, 10 μ M)	0.5μL
Recombinant ribonuclease inhibitor(Takara)	0.5μL
		T4 RNA Ligase 1(ssRNA Ligase)(NEB)	1μL
ATP(10mM)(NEB)	1μL

The reaction was incubated at 25 ℃ for 2 hours.

3) Reverse transcription to synthesize cDNA

The reaction system for reverse transcription is shown in the following table:

IP or Input ligation product in step 2)	20μL
		Reverse transcription primer (RTPrimer) (10. mu.M)	1μL
10×RT Mix(Vazyme)	3μL
		HiScript III Enzyme Mix(Vazyme)	3μL
RNase-free water	3μL

The reaction conditions for the reverse transcription are shown in the following table:

50℃	1 hour
		85℃	1 minute
4℃	-

4) PCR amplification and purification

The reaction system for PCR amplification is shown in the following table:

the reaction conditions for PCR amplification are shown in the following table:

after the reaction is finished, 1 XVAHTS DNA clean beads are used for purification, 30 mu L of purified IP and Input libraries are obtained respectively, the libraries are sent to Annuodda gene technology limited company for sequencing after the library concentration (more than 4 ng/mu L) and the fragment size (200-500bp) are detected to be qualified, and the high-throughput sequencing is carried out on a HiSeqX sequencing platform.

(6) Data analysis and results

And downloading a sequencing result, and performing data analysis. Firstly, according to the corresponding relation between the sample and the barcode, fastq-multx (https:// github. com/brwnj/fastq-multx) is used for splitting the mixed sample data. The raw sequencing data was then quality evaluated using FastQC (http:// www.bioinformatics.babraham.ac.uk/projects/FastQC /), using Cutadapt (https:// cutapto. readthe docs. io/en/stable /) to remove linker sequences as well as low quality data, using fastp (https:// github. com/OpenGene/fastp) to remove terminal index and barrel sequences, obtaining clean data that was ultimately used for downstream analysis and quality evaluation again. The sequencing fragments were aligned to the reference genome using Hisat2(https:// daehwankimlab. github. io/Hisat2/), and aligned filtered, compressed, sorted and indexed using samtools (https:// github. com/samtools/samtools) to obtain the bam file. PeakCalling was performed using Macs2(https:// github. com/Macs3-project/MACS) to identify the m6A site and obtain the peak file. Differential methylation analysis was performed using DEQ (https:// githu. com/al-mcinntyre/DEQ), identifying regions of differential methylation between samples and calculating fold differential enrichment, enabling relative quantification of m6A between samples.

The current research shows that most of m6A modifications are enriched near a stop codon, and most of the technical development research uses the distribution mode as the basis for judging the feasibility of the method. In this example, the mixed results of two RNA samples of mES wild-type and 293T cells were analyzed, and the distribution is shown in FIG. 2, wherein m6A of both samples is significantly enriched near the stop codon, which is consistent with the current research results. The motif analysis results are shown in FIG. 3, and the top ranked results show the typical "RRACH" (R ═ G or A; H ═ A, C, or U) motif of m6A, which meets the known conclusions (references: dominisini D, Moshitch-Moshkovitz S, Schwartz S, et al. polarity of the human and motor m6A RNA methyl modified by m6A-seq [ J ]. Nature,2012,485(7397):201 and 206.). Example 2A method for mixed sample detection of m6A modification by Barcode labeling

(1) oligo Barcode labeling and Synthesis of Universal primers

1) Synthesis of Oligo Barcode tags (i.e., Barcode-containing 3' end linkers)

2) the synthesis of the universal primer, the details of the specific sequence and the sequence characteristics are the same as those of example 1.

(2) Adenylation treatment of end connector containing barcode3

end connector containing barcode 3': RLA3(NEB) -Barcode 1/2/3/4	150pmol
		10×5'DNA Adenylation Reaction Buffer(NEB)	2μL
ATP(1mM)(NEB)	2μL
		Mth RNA ligase(NEB)	2μL
RNase-free water	To 20 μL

65℃	1 hour
		85℃	5 minutes

(3) RNA fragmentation

Selecting four RNA samples of mES wild type RNA, Wtap knockout RNA, Mettl3 knockout RNA and Mettl14 knockout RNA (RNA samples with rRNA removed, derived from mouse cells and purchased from cell banks of Chinese academy of sciences), and respectively carrying out fragmentation treatment on each RNA sample, wherein the reaction system of single fragmentation is shown in the following table:

(4) End repair of fragmentation products

the reaction conditions for a single repair are shown in the table below:

37℃	30 minutes
		65℃	10 minutes
4℃	-

(5) Barcode labeling of different samples

And (3) respectively connecting each mRNA sample repaired in the step (4) with an adenylated Barcode-containing 3 'end connector, wherein the connection reaction system of a single-time 3' end connector (containing Barcode) is shown as the following table (namely, mES wild type mRNA, Wtap knockout type mRNA, Mettl3 knockout type mRNA and Mettl14 knockout type mRNA are respectively marked by adopting the adenylated Barcode 1-4):

the reaction was incubated at 25 ℃ for 2 hours and then at 4 ℃ overnight.

30℃	30 minutes
		37℃	30 minutes
70℃	5 minutes

After the reaction is finished, all the Barcode-labeled RNA samples are mixed, purified by using RNA clean & concentrator-5(Zymo Research), 20 mu L of RNase-free water is taken to elute all the products, and the concentration is detected to ensure that the total amount of each Barcode-labeled RNA sample is more than 500ng (in the case of shortage, a proper amount of RNAscope-in can be added).

(6) Mixed sample MeRIP: the same as in example 1.

(7) Data analysis and results: the data analysis was the same as in example 1. The experimental results are as follows:

previous studies have shown that the dynamic change process of m6A in organisms is regulated by various proteins such as methyltransferases, demethylases, m6A recognition proteins, etc., among which the proteins involved in the methyl transfer process of m6A are known as WTAP, METTL3 and METTL 14. In this example, M6A sites in mouse Wild Type (WT), Wtap knock-out (WTAPKO), Mettl3 knock-out (M3KO), Mettl14 knock-out (M14KO) cell samples were detected and analyzed, and the distribution of WT was shown in fig. 4, which is similar to known results, and M6A is significantly enriched near the stop codon. For the Wtap and Mettl3 knock-out samples, the enrichment near the stop codon was significantly reduced, whereas the Mettl14 knock-out sample did not change significantly. Motif analysis results As shown in FIG. 5, WT samples had a distinct "RRACH" (R ═ G or A; H ═ A, C, orU) pattern, consistent with the known conclusions (references: Meyer, K.D., Saletore, Y., Zumbo, P., Elemento, O., Mason, C.E., and Jaffrey, S.R. (2012). Compressent analysis of mRNA methylation reactions in 3' UTRs and near stop codes.cell 149, 1635-1646). Quantitative difference analysis results of WT and M3KO samples as shown in fig. 6, M6A levels of the Mettl3 knockout sample were significantly reduced near the stop codon compared to WT.

It can be seen from the comprehensive examples 1 and 2 that the Barcode label is adopted to perform sample mixing detection m6A modification, so that not only can the m6A modification condition in RNA be effectively monitored, but also the input amount of a single sample can be greatly reduced, the accurate quantification of the input amount of the sample can be realized, the situation that MeRIP-seq cannot be detected due to too low sample amount can be avoided, and meanwhile, errors caused by different experimenters and antibody batch effects can be avoided.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Sequence listing

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Claims

1. A method for detecting RNA modification and realizing accurate quantification by mixed sample is characterized by comprising the following steps:

s1 synthesizing a product containing a Barcode sequence and having NH at the 3' end₂C6 modified and 3 'end connector with phosphate modification at 5' end, and carrying out 5 'end adenylation on the 3' end connectorTreating to facilitate subsequent ligation reactions;

2. The method for mixed sample detection of RNA modification and accurate quantification as claimed in claim 1, wherein the Barcode sequence is 6 base Barcode or 2 random bases +6 base Barcode.

3. The method for mixed sample detection of RNA modification and accurate quantification as claimed in claim 1, wherein the 3' terminal linker comprises RLA3(NEB) -Barcode1 shown in SEQ ID NO.1, RLA3(NEB) -Barcode2 shown in SEQ ID NO.2, RLA3(NEB) -Barcode3 shown in SEQ ID NO.3, and RLA3(NEB) -Barcode4 shown in SEQ ID NO. 4.

4. The method for mixed sample detection of RNA modification and accurate quantification as claimed in claim 1, wherein fragmentation and end repair of RNA samples are required before the end linker containing Barcode3 'after adenylation is connected to the 3' ends of different RNA samples.

5. The method for detecting RNA modification and achieving precise quantification of claim 1, wherein step S2 is to use KQ-mutated T4RNA ligase to ligate an adenylated end linker containing Barcode3 'to the 3' end of the RNA sample.

6. The method of claim 1, wherein step S4 is a step of detecting RNA modification and performing precise quantificationUsing a 5' terminal NH₂C6 modified 5 'linker was ligated to the 5' end of RNA fragment by T4RNALIGAse1, followed by reverse transcription using universal primers and finally library amplification without purification.

7. The method for mixed sample detection of RNA modification and accurate quantification as claimed in claim 6, wherein the sequence of the 5' linker is shown as SEQ ID No.5, and the sequence of the universal primer is shown as SEQ ID No. 6.

8. The method of claim 1, wherein in step S4, the data analysis comprises splitting the mixed sample data by Barcode sequence, comparing the RNA samples, identifying peakcalling and m6A locus, and finally performing differential methylation analysis by combining the results of the samples.