CN115491414A - Comprehensive analysis method for methylation modification differential expression of rheumatoid arthritis complete transcriptome m6A - Google Patents

Abstract

The invention relates to a comprehensive analysis method for methylation modification differential expression of a complete transcriptome m6A of rheumatoid arthritis. The invention adopts high-throughput sequencing combined with methylated RNA immunoprecipitation and RNA sequencing to obtain the m6A modification of the total transcriptome of the synovial membrane of the RA patient, and further enriches downstream signal channels. 2025 transcripts in RA synovium were found to have differential methylation sites, of which 1016 peaks were significantly up-regulated and 1009 peaks were significantly down-regulated; combined analysis of m6A-seq and RNA-seq showed that 120 genes in total had significant changes in both m6A modification and mRNA expression; GO and KEGG analysis shows that the m6A gene with differential expression is closely related to immune reaction, vascular endothelium, MAPK signal path, PI3K-Akt signal and other inflammatory processes involved in macrophage activation.

Description

Comprehensive analysis method for methylation modification differential expression of rheumatoid arthritis complete transcriptome m6A

Technical Field

The invention belongs to the field of molecular biology of rheumatoid diseases, and particularly relates to a comprehensive analysis method for methylation modification differential expression of a complete transcriptome m6A of rheumatoid arthritis, and specifically relates to application of a methylation modification differential expression gene of the complete transcriptome m6A in research of progression and pathogenesis of rheumatoid arthritis.

Background

Rheumatoid Arthritis (RA) is an immune-mediated chronic disease with synovial involvement as the major component. The specific pathogenesis is not completely understood at present, and the immunity, life style, heredity and environmental factors are related to the pathogenesis. However, there is no good explanation as to how the interaction between factors plays a role in the pathogenesis of RA. The ambiguity of the pathogenesis of RA brings difficulty for accurate early diagnosis and treatment, and research methods for exploring the pathogenesis of RA are urgently needed.

Epigenetic factors may become important mediators linking genetics and gene expression for explaining the cause of RA pathogenesis. The role of epigenetic, particularly RNA modification in arthritis, is of great interest. In recent years, more and more research has begun to focus on the role of epigenetics in the pathogenesis of RA. Epigenetic regulation allows genetic changes in gene function without changing any DNA sequence, ultimately resulting in altered disease phenotypes including RNA methylation, DNA methylation, covalent histone modification, and the like. Epigenetic modifications of DNA and histones have been shown to be involved in the pathogenesis of rheumatoid arthritis, but less research has been conducted to determine whether modifications involving N6-methyladenosine (m 6A) in RNA are involved.

m6A methylation plays an important role in a variety of biological processes by controlling gene expression, including immune recognition, activation of innate and adaptive immune responses, and important regulatory effects on immunity, viral infection, and autoinflammatory diseases. m6A methylation modification is involved in the process of rheumatoid arthritis disease and plays an important role in RA disease progression, treatment and prognosis. The differential expression genes of the RA whole transcriptome are explored from the m6A perspective and the effects of the genes are researched, and the comprehensive understanding of the m6A modified mRNAs participating in the attack of RA is helpful for understanding the role of epigenetics in RA and for searching new therapeutic targets.

Disclosure of Invention

Aiming at the modification research of m6A in RNA lacking in RA pathogenesis at present, the invention provides a comprehensive analysis method for methylation modification differential expression of m6A in a rheumatoid arthritis complete transcriptome, which comprises the following specific steps:

(1) and RNA extraction:

1) Collecting synovial tissue, and adding 1ml of TRIzol for cracking;

2) After the cracking is completed, adding 0.2mL of chloroform, violently shaking for 15 seconds, and standing for 5 minutes at room temperature;

3) Centrifuging at 12000rpm for 10 min at 4 ℃, taking the supernatant and adding into another EP tube;

4) Adding 0.5mL of precooled isopropanol, gently mixing uniformly, and incubating for 30 minutes at-20 ℃;

5) Centrifuging at 12000rpm at 4 deg.C for 15 min, and discarding the supernatant;

6) 1mL of precooled 75% ethanol was added, and the absolute ethanol was diluted with DEPC water; centrifuging at 12000rpm at 4 deg.C for 5 min, and discarding the supernatant;

7) Repeating step 6);

8) Drying the RNA precipitate at room temperature, adding 20-50 μ L DEPC water, and storing at-80 deg.C;

(2) total RNA quality detection

(1) Preparing 100ml of DEPC water for diluting the concentration of RNA and washing the cuvette;

(2) Preparing a filter paper sheet and a filter paper strip for sucking residual liquid in the cuvette;

(3) Taking out the sample, putting the sample into an ice box, sucking 100 mul DEPC water, putting the sample into a measuring instrument, pressing a blank key, and zeroing;

(4) Sucking 1 mul of sample, and repeatedly sucking by using a 100 mul gun head to fully and uniformly mix the sample and water;

(5) Measuring the concentration, recording the data (R value: OD260/280, OD260/230 concentration);

(6) 1.8 sP R >;

(3) rRNA removal, RNA purification and fragmentation

Taking a proper amount of total RNA to remove rRNA from the total RNA, incubating the total RNA with an rRNA probe aiming at a specific species to capture the rRNA in the total RNA, wherein the probe is modified by biotin, and combining magnetic beads coated with streptavidin with the probe and an rRNA compound to remove the rRNA; then obtaining RNA without rRNA, including mRNA with polyA and without polyA, non-coding RNA and regulating RNA after Magicpure RNA Beads magnetic bead purification; fragmenting rRNA-removal in the presence of metal ions to obtain RNA fragments with the length range of 60-200 bp; reserving RNA with the volume of 1/10 as Input, and carrying out next antibody immunoprecipitation on other RNA;

(4) antibody immunoprecipitation

Mixing Dynabeads Protein A magnetic beads and antibodies in advance, and performing rotary incubation for 2 hours at 4 ℃ to obtain a compound of the magnetic beads and the antibodies; adding fragmented RNA into the magnetic bead-antibody solution, and rotationally incubating at 4 ℃ for 2h to combine the RNA fragment with m6A methylation modification with the antibody; separating the magnetic beads from the solution by using a magnetic frame, removing the solution, adding a wash buffer to resuspend the magnetic beads, fully washing for 3-5 times, removing RNA fragments non-specifically bound with the magnetic beads, and improving the sensitivity of an experimental result; adding m 6A-containing eluent into the magnetic beads without non-specifically bound RNA, and competitively eluting the m 6A-modified RNA fragments with specific binding, wherein the RNA fragments are marked as IP;

(5) strand-specific library construction

1) Reverse transcription to synthesize single-strand cDNA

Synthesizing first strand cDNA by using IP and Input RNA as templates under the action of a first strand synthesis buffer solution and Invitrogen reverse transcriptase SuperScript IV; meanwhile, actinomycin D is added into the one-strand synthesis buffer solution, so that the false reverse transcription depending on a DNA template is prevented, and the reverse transcription specificity with RNA as the template is ensured;

2) Synthesis of second Strand cDNA

Adding a double-strand synthesis premixed system into a system for synthesizing single-strand cDNA, firstly hydrolyzing and digesting RNA on an RNA/cDNA hybrid strand, then synthesizing double-strand cDNA with dUTP by using DNA polymerase with the single-strand cDNA as a template and dNTP Mix replacing dTTP by dUTP as a substrate; in the subsequent PCR amplification step, the second strand cDNA randomly doped with dUTP cannot be identified and amplified by the high-fidelity polymerase, so that only library information from the first strand cDNA is amplified, and the construction of a strand-specific library is realized; after one-time MagicPure DNA Beads II magnetic bead purification, finally obtaining double-stranded cDNA capable of directly adding A at two ends;

3) Reaction of 3' end with "A

Adding an A buffer reaction system to the 3' end of the system; adding single adenylic acid A at the 3 'ends of the two sides of the double-stranded cDNA with the modified ends to prevent the self-connection of the flat ends among the cDNA fragments, and the single adenylic acid A can be complementary matched with the single T at the 5' end of the sequencing joint in the next step to accurately connect, thereby effectively reducing the self-connection among the library fragments;

4) Ligation sequencing adapter

Adding a connecting buffer solution and a double-strand sequencing joint into the reaction system, and connecting the illumina sequencing joint to the two ends of the library DNA by using T4 DNA ligase; incubation is carried out for 10 minutes at 30 ℃, and the ligation reaction is rapidly and effectively carried out;

5) Library fragment screening

For the library added with the joint, the MagicPure DNA Beads II nucleic acid fragment screening kit is used for screening the size of the fragment while purifying the library; screening by adopting a two-step method, firstly removing small fragments on the left side of a target region by using SPRI magnetic beads, then removing large fragments on the right side of the target region, and finally screening an original library with the fragment peak value of 250bp for the next PCR amplification; the library after purification removes excessive sequencing joints in the system and products of joint self-connection, avoids ineffective amplification in the PCR amplification process, and eliminates the influence on-machine sequencing;

6) PCR amplification of cDNA libraries

Amplifying the original library in a 50 μ L reaction system using high fidelity polymerase to ensure sufficient library inventory on the sequencer; only DNA fragments with joints on both ends can be amplified, and only the fragments connected with single-ended joints are removed, so that the qualified library is enriched; meanwhile, the newly generated double-strand cDNA is provided with dUTP, so that the amplification of high-fidelity polymerase using the dUTP as a template is prevented, the amplified library is only derived from the single-strand cDNA, and the strand direction information is faithfully reserved; the number of PCR amplification cycles is controlled between 12 and 15; on the premise of ensuring that the product is sufficient, the deviation caused by overlarge amplification cycle number is reduced; purifying the amplified library by magnetic beads to obtain a sequencing library which can be operated on a computer;

7) Library quality testing

Performing quality detection and quantification on the constructed sequencing library; accurately quantifying the concentration of the library by using the Qubit, determining the size distribution of the fragments of the library by using an Agilent 2100Bioanalyzer, and evaluating whether the library is suitable for being installed on a computer;

8) Sequencing on computer

Diluting samples qualified for quality inspection, and mixing and loading a plurality of samples according to corresponding molar proportions according to the sequencing flux requirements of different samples; sequencing the library by adopting an Illumina high-throughput sequencing platform and a 2 x 150bp double-end sequencing strategy;

(6) data analysis

1) mRNA raw data quality assessment

2) Statistics of sequencing data volume

3) Base mass distribution of raw data

4) Raw data filtering

5) Reference gene alignment

6) Quantitative expression of mRNA

7) Differential expression analysis of mRNA

8) Enrichment analysis of differentially expressed genes

9) Methylation site prediction

10 Analysis of methylation site differences

11 Analysis of methylation differential site Gene enrichment

12 Combined analysis of methylation differential site genes with differentially expressed genes.

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

the invention systematically and comprehensively analyzes the differential expression of RA whole transcriptome m6A methylation modification, and the research of the embodiment proves that: there were 2025 transcripts with differential methylation sites (peaks) in the RA synovium, of which 1016 peaks were significantly upregulated. 1009 peaks were significantly down-regulated. GO and KEGG analysis shows that the m6A gene with differential expression is closely related to the inflammatory processes of macrophage activation participating in immune reaction, vascular endothelium, MAPK signal path, PI3K-Akt signal path and the like. Furthermore, a total of 120 genes showed significant changes in both m6A modification and mRNA expression by combined analysis of m6A-seq and RNA-seq. The discovery can hopefully further enrich and perfect the research of the mechanism of RAM6A methylation modified RNA, and also bring hopes for developing novel biomarkers for RA early diagnosis, targeted therapy and prognosis evaluation.

Drawings

FIGS. 1A-E show the analysis of the transcriptome information for RA synovium differentially expressed mRNA.

FIGS. 2A-E show the distribution of m6A modified epigenetic transcriptomes.

FIGS. 3A-D show the m6A modified epigenet KEGG, GO analysis.

FIGS. 4A-F show RNA sequencing vs MeRIP sequencing analyses.

Detailed Description

The present invention will be further described with reference to specific embodiments, which are provided for the purpose of illustrating the principles and procedures of the present invention and are not to be construed as limiting the invention. Those skilled in the art will appreciate that various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention.

The following examples are examples of experimental methods in which specific conditions are not specified, and the tests are usually carried out under the conventional conditions or under the conditions recommended by the manufacturers.

Example 1

The invention provides a comprehensive analysis method for methylation modification differential expression of a complete transcriptome m6A of rheumatoid arthritis, which comprises the following specific steps:

(1) and RNA extraction:

the sequencing sample information is shown in table 1.

TABLE 1 sequencing sample information

1) Synovial tissue was collected and lysed by adding 1ml TRIzol.

2) After completion of the lysis, 0.2mL of chloroform was added, followed by vigorous shaking for 15 seconds, and the mixture was left at room temperature for 5 minutes.

3) Centrifuge at 12000rpm for 10 minutes at 4 ℃ and take the supernatant (approximately 500 ul) and add to another EP tube.

4) 0.5mL of pre-cooled isopropanol was added, gently mixed and incubated at-20 ℃ for 30 minutes.

5) After centrifugation at 12000rpm at 4 ℃ for 15 minutes, the supernatant was discarded.

6) Add 1mL precooled 75% ethanol (absolute ethanol with DEPC water dilution). Centrifuge at 12000rpm for 5 minutes at 4 ℃ and discard the supernatant.

7) Repeat step 6).

8) The RNA precipitate was dried at room temperature, 20-50. Mu.L of DEPC water was added, and the mixture was stored at-80 ℃ for further use.

(2) Total RNA quality detection

(1) 100ml of DEPC water was prepared to dilute the RNA concentration and wash the cuvette.

(2) A piece of filter paper and a strip of filter paper were prepared to suck up the remaining liquid in the cuvette.

(3) The sample was taken out and placed in an ice box, 100. Mu.l of DEPC water was aspirated and placed in the measuring instrument, and the blank key was pressed and zeroed.

(4) Aspirate 1. Mu.l of the sample and mix the sample with water by pipetting repeatedly with a 100. Mu.l pipette tip.

(5) The concentration was measured and the data were recorded (R value: OD260/280, OD260/230 concentration).

(6) 1.8 sR > -2.0, indicating that the total RNA quality is satisfactory.

(3) rRNA removal, RNA purification and fragmentation

Taking a proper amount of total RNA to remove rRNA, incubating the total RNA with an rRNA probe aiming at a specific species (Human/Mouse/Rat), capturing the rRNA in the rRNA, modifying the probe with Biotin (Biotin), and combining the probe and an rRNA compound by using magnetic beads coated with streptavidin to remove the rRNA. Then, after the magnetic bead purification of the MagicPure RNA Beads, the rRNA-removed RNA (rRNA-remove) is obtained, and the RNA comprises mRNA with polyA and without polyA, non-coding RNA and regulating RNA. rRNA-removal is fragmented in the presence of metal ions to obtain RNA fragments with a length range of 60-200 bp. 1/10 volume of RNA was retained as Input, and the other RNAs were subjected to the next antibody immunoprecipitation.

(4) Antibody immunoprecipitation

Dynabeads Protein A magnetic beads and antibodies are mixed in advance, and the mixture is incubated for 2 hours at 4 ℃ in a rotating mode to obtain a compound of the magnetic beads and the antibodies. Fragmented RNA was added to the bead-antibody solution and incubated at 4 ℃ for 2h with rotation to allow binding of the m6A methylated RNA fragments to the antibody. And separating the magnetic beads from the solution by using a magnetic frame, removing the solution, adding a wash buffer to resuspend the magnetic beads, fully washing for 3-5 times, removing RNA fragments which are not specifically combined with the magnetic beads, and improving the sensitivity of an experimental result. And adding an m 6A-containing eluent into the magnetic beads from which the non-specifically bound RNA is removed, and competitively eluting the specifically bound m 6A-modified RNA fragments, which are marked as IP.

(5) Strand-specific library construction

1) Reverse transcription to synthesize single-stranded cDNA

Synthesizing first strand cDNA by using IP and Input RNA as templates under the action of a first strand synthesis buffer solution and Invitrogen reverse transcriptase SuperScript IV (SS IV); meanwhile, actinomycin D is added into the one-strand synthesis buffer solution, so that the false reverse transcription depending on a DNA template is prevented, and the reverse transcription specificity with RNA as the template is ensured.

2) Synthesis of second Strand cDNA

Adding a double-strand synthesis premixed system into a system for synthesizing single-strand cDNA, reacting, firstly, hydrolyzing and digesting RNA on an RNA/cDNA heterozygous strand, then, synthesizing double-strand cDNA with dUTP by using DNA polymerase with the single-strand cDNA as a template and dNTP Mix replacing dTTP by dUTP as a substrate; in the subsequent PCR amplification step, the second strand cDNA randomly doped with dUTP cannot be identified and amplified by high-fidelity polymerase, so that only library information derived from one strand cDNA is amplified, and the construction of a strand-specific library is realized; after one-time MagicPure DNA Beads II magnetic bead purification, double-stranded cDNA (Double-strand cDNA, ds cDNA) with 'A' directly added at both ends is finally obtained.

3) Reaction of 3' end with "A

To the above system was added an "A" buffer reaction system at the 3' end. And adding single adenosine A at the 3 'ends of both sides of the double-stranded cDNA after the end modification to prevent the blunt end self-connection between cDNA fragments, and the single adenosine A can be matched with the single T protrusion at the 5' end of the sequencing adaptor in the next step in a complementary way to be accurately connected, thereby effectively reducing the self-series connection between the library fragments.

4) Ligation sequencing adapter

And adding a connecting buffer solution and a double-strand sequencing joint into the reaction system, and connecting the illumina sequencing joint to the two ends of the library DNA by using T4 DNA ligase. Incubation is carried out for 10 minutes at 30 ℃ to carry out the ligation reaction rapidly and effectively.

5) Library fragment screening

For the library with the linker, fragment size screening was performed while purifying the library using the MagicPure DNA Beads II nucleic acid fragment screening kit. A two-step screening (Double Size selection) is adopted, a small fragment (Left-side Size selection) on the Left side of a target region is removed by SPRI magnetic beads, a large fragment (Right-side Size selection) on the Right side of the target region is removed, and an original library with a fragment peak value of 250bp is finally screened and used for the next PCR amplification. The purified library eliminates excessive sequencing joints in the system and products of self-connection of the joints, avoids ineffective amplification in the PCR amplification process and eliminates the influence on-machine sequencing.

6) PCR amplification of cDNA libraries

The original library was amplified using high fidelity polymerase in a 50. Mu.L reaction to ensure sufficient library inventory on the sequencer. Only DNA fragments with joints at both ends can be amplified, and the fragments only connected with single-ended joints are removed, so that the qualified library is enriched; meanwhile, the newly generated double-strand cDNA is provided with dUTP, so that the amplification of a high-fidelity polymerase using the dUTP as a template is prevented, the amplified library is only derived from the single-strand cDNA, and the strand direction information is faithfully preserved. The number of PCR amplification cycles was controlled between 12 and 15. On the premise of ensuring enough products, the deviation caused by the excessive number of amplification cycles is reduced. And purifying the amplified library by magnetic beads to obtain the sequencing library which can be operated on a computer.

7) Library quality detection

And (4) performing quality inspection and quantification on the constructed sequencing library. The library concentration was accurately quantified using Qubit, the size distribution of the library fragments was determined using the Agilent 2100Bioanalyzer, and whether the assay was suitable for loading.

8) Sequencing on computer

And diluting the qualified samples, and mixing and loading a plurality of samples according to corresponding molar proportions according to the sequencing flux requirements of different samples. The library was sequenced using the Illumina high-throughput sequencing platform with a 2 × 150bp paired-end sequencing strategy.

(6) Data analysis

1) mRNA raw data quality assessment

2) Statistics of sequencing data volume

3) Base mass distribution of raw data

4) Raw data filtering

5) Reference gene alignment

6) Quantitative expression of mRNA

7) Differential expression analysis of mRNA

8) Enrichment analysis of differentially expressed genes

9) Methylation site prediction

10 Analysis of methylation site differences

11 Methylation differential site Gene enrichment analysis

12 A combination of methylation differential site genes and differentially expressed genes.

Example 2

And (3) result characterization:

1. and (3) analyzing the transcriptome information of RA synovial membrane differential expression mRNA.

RA synovial membrane RNA transcriptome information was analyzed using RNA high throughput sequencing. The distribution of differentially expressed genes in the RA group compared to the HC group showed that the amount of upregulated mRNA was 463. The number of downregulated mrnas was 603. The genes which are up-regulated and distributed are respectively ANGPTL7, CLSTN2, MFAP5 and the like, and the genes which are down-regulated and distributed are respectively ADAM12, COL5A3, TOP2A and the like (see figure 1A).

GO and KEGG. Up-regulated genes are mainly enriched in the regulation of vascular definition, muscle cell promotion, extracellular matrix, etc. The signal pathway of differential gene enrichment is related to MAPK, adipocyte signaling pathway (see FIGS. 1B-1C).

The down-regulated genes are mainly enriched in platelet-derived growth factor binding, collagen-containing extracellular matrix and the like. The signaling pathway is related to PI3K-Akt, IL-17signaling pathway (see FIGS. 1D-1E).

2. m6A modifies the apparent transcript profile.

2025 transcripts in RA synovium showed differential methylation sites (peaks) compared to HC. Of these, 1016 peaks were significantly upregulated. 1009 peaks were significantly downregulated (Fold change >2.0 and P < 0.05). In the RA synovial m6A modified transcriptome, 2025 transcripts corresponded to 1754 genes in total. The differential peak of m6A modification of each sample was distributed predominantly in the 3 'untranslated region (3' utr) and adjacent coding region (CDS terminus) (see figure 2a, 2b).

Multiple m6A modification sites are visible on the same transcript. More than half of the methylation modification sites are located in the 3' UTR region. For transcripts with peak numbers greater than 1, the site of methylation modification was predominantly located in the CDS region (see FIG. 2C). Consensus sequences for motif enrichment analysis are shown in FIG. 2D.

The genes corresponding to transcripts with differential methylation modification are widely distributed on chromosomes 1, 2, 3, 11, 17, and 19 (number of m6A peaks > 100) (see FIG. 2E).

3. m6A modified epigenome KEGG, GO analysis.

To further reveal the m6A modified epigenetic transcriptome information. Screening results of MeRIP sequencing. Obtaining the enrichment item of the gene corresponding to the differential methylation modified transcript. As shown in fig. 3A and 3B.

The main enriched items of the genes corresponding to the methylation modification up-regulated transcript are cytosolic and endosomastransport, regulation of cell morphology, cell-cell signaling by wnt and the like.

Methylation modification Down-Regulation of major enrichment items are macrophage activation inactivated in immune response, MAP kinase tyrosine/serum/threonine phosphate activity, and the like.

KEGG lists top 10 pathway information corresponding to gene enrichment in transcripts with significant m6A modification, respectively. The KEGG signaling pathway shows that methylation modification is upregulated mainly in MAPK, wnt signaling pathway, etc. Genes corresponding to down-regulated transcripts were mainly enriched in pathways such as mRNA surveyability pathway, hippo signaling pathway, etc. (see FIGS. 3C and 3D).

4. RNA sequencing was analyzed in conjunction with merrip sequencing.

To reveal the regulatory relationship of mRNA m6A modification to its transcriptional level. MeRIP-seq and RNA-seq data were correlated. 120 genes were found to contain significantly different m6A modification peaks and expression changes (see FIG. 4A).

There were 47 transcripts up-regulated by methylation modification. There were 73 transcripts downregulated by methylation modification. Of the 47 transcripts up-regulated by methylation modification, 18 transcripts were up-regulated in expression simultaneously. 29 reverse downregulations. Of the 73 transcripts that were downregulated by methylation modification, 34 transcripts were downregulated simultaneously. 39 are turned upwards. As shown in fig. 4B.

And (3) analyzing and displaying the intersection genes GO and KEGG of the differential expression genes and the m6A peak. The mRNA with the transcript expression synchronous up-regulation difference is mainly enriched in mRNA methyltransferase activity, apoptosis-multiple species, cytokine receptor activity and the like. The signal paths are PI3K-Akt signaling pathway, JAK-STAT signaling pathway and the like (see figures 4C and 4D).

The simultaneous downregulation of differential mRNA by transcript expression was mainly enriched in the muscle tissue level, extracellular matrix, etc. (see FIGS. 4E, 4F).

And fifthly, methylation modification of the transcript gene expression of the RA patient.

And performing correlation analysis by combining the signal path information enriched by MeRIP-seq data and the result of correlation analysis of RNA sequencing and MeRIP sequencing.

As a result, it was found that mRNA at the first 3 positions was up-and down-regulated in RA patients, and a total of 12 genes were obtained (see Table 2). Specifically PTEN, ASPM, SHCBP1, LDLR, NDRG2, ITIH5, IL17RD, DYSF, RELN, ZBTB16, NXPH3, and SEMA4A. In these 12 genes, the methylation-modified transcripts were up-regulated simultaneously to ZBTB16, NXPH3, SEMA4A. The methylated modified transcripts are synchronously down-regulated to PTEN, ASPM, SHCBP1, LDLR.

TABLE 2 differential methylation site Gene information of mRNA differential expression and MeRIP obtained by RNA sequencing of RA patients

The foregoing is merely exemplary and illustrative of the principles of the present invention and various modifications, additions and substitutions of the specific embodiments described herein may be made by those skilled in the art without departing from the principles of the present invention or exceeding the scope of the claims set forth herein.

Claims (1)

1. The comprehensive analysis method for methylation modification differential expression of the rheumatoid arthritis complete transcriptome m6A is characterized by comprising the following steps of:

(1) and RNA extraction:

1) Collecting synovial tissue, and adding 1ml of TRIzol for cracking;

2) After the cracking is completed, adding 0.2mL of chloroform, violently shaking for 15 seconds, and standing for 5 minutes at room temperature;

3) Centrifuging at 12000rpm at 4 deg.C for 10 min, and adding the supernatant into another EP tube;

4) Adding 0.5mL of precooled isopropanol, gently mixing uniformly, and incubating for 30 minutes at-20 ℃;

5) Centrifuging at 12000rpm at 4 deg.C for 15 min, and discarding the supernatant;

6) Adding 1mL of precooled 75% ethanol, and diluting absolute ethanol with DEPC water; centrifuging at 12000rpm at 4 deg.C for 5 min, and discarding the supernatant;

7) Repeating step 6);

8) Drying RNA precipitate at room temperature, adding 20-50 μ L DEPC water, and storing at-80 deg.C;

(2) total RNA quality detection

(1) Preparing 100ml of DEPC water for diluting the RNA concentration and washing the cuvette;

(2) Preparing a filter paper sheet and a filter paper strip for sucking residual liquid in the cuvette;

(3) Taking out the sample, putting the sample into an ice box, sucking 100 mu l of DEPC water, putting the DEPC water into a measuring instrument, pressing a blank key, and zeroing;

(4) Sucking 1 mul of sample, and repeatedly sucking by using a 100 mul gun head to fully and uniformly mix the sample and water;

(5) Measuring the concentration, recording the data (R value: OD260/280, OD260/230 concentration);

(6) 1.8 sP R >;

(3) rRNA removal, RNA purification and fragmentation

Taking a proper amount of total RNA to remove rRNA from the total RNA, incubating the total RNA with an rRNA probe aiming at a specific species to capture the rRNA in the total RNA, wherein the probe is modified by biotin, and combining magnetic beads coated with streptavidin with the probe and an rRNA compound to remove the rRNA; then obtaining RNA without rRNA, including mRNA with polyA and without polyA, non-coding RNA and regulating RNA after the magnetic bead purification of the Magicpure RNA; fragmenting rRNA-removal in the presence of metal ions to obtain an RNA fragment with the length range of 60-200 bp; reserving 1/10 volume of RNA as Input, and carrying out next antibody immunoprecipitation on other RNA;

(4) antibody immunoprecipitation

Mixing Dynabeads Protein A magnetic beads and antibodies in advance, and carrying out rotary incubation for 2h at 4 ℃ to obtain a compound of the magnetic beads and the antibodies; adding fragmented RNA into the magnetic bead-antibody solution, and rotationally incubating at 4 ℃ for 2h to combine the RNA fragment with m6A methylation modification with the antibody; separating the magnetic beads from the solution by using a magnetic frame, removing the solution, adding a wash buffer to resuspend the magnetic beads, fully washing for 3-5 times, removing RNA fragments which are not specifically combined with the magnetic beads, and improving the sensitivity of an experimental result; adding m 6A-containing eluent into the magnetic beads without non-specifically bound RNA, and competitively eluting the m 6A-modified RNA fragments with specific binding, wherein the RNA fragments are marked as IP;

(5) strand-specific library construction

1) Reverse transcription to synthesize single-strand cDNA

Synthesizing first strand cDNA by using IP and Input RNA as templates under the action of a first strand synthesis buffer solution and Invitrogen reverse transcriptase SuperScript IV; meanwhile, actinomycin D is added into the one-strand synthesis buffer solution, so that the false reverse transcription depending on a DNA template is prevented, and the reverse transcription specificity with RNA as the template is ensured;

2) Synthesis of second Strand cDNA

Adding a double-strand synthesis premixed system into a system for synthesizing single-strand cDNA, reacting, firstly, hydrolyzing and digesting RNA on an RNA/cDNA heterozygous strand, then, synthesizing double-strand cDNA with dUTP by using DNA polymerase with the single-strand cDNA as a template and dNTP Mix replacing dTTP by dUTP as a substrate; in the subsequent PCR amplification step, the second strand cDNA randomly doped with dUTP cannot be identified and amplified by the high-fidelity polymerase, so that only library information from the first strand cDNA is amplified, and the construction of a strand-specific library is realized; after one-time MagicPure DNA Beads II magnetic bead purification, finally obtaining double-stranded cDNA which can be directly added with 'A' at two ends;

3) Reaction of 3' end with "A

Adding an A buffer reaction system at the 3' end into the system; adding single adenylic acid A at the 3 'ends of the two sides of the double-stranded cDNA with the modified ends to prevent the self-connection of the flat ends among the cDNA fragments, and the single adenylic acid A can be complementary matched with the single T at the 5' end of the sequencing joint in the next step to accurately connect, thereby effectively reducing the self-connection among the library fragments;

4) Ligation sequencing adapter

Adding a connecting buffer solution and a double-strand sequencing joint into the reaction system, and connecting the illumina sequencing joint to the two ends of the library DNA by using T4 DNA ligase; incubating for 10 minutes at 30 ℃ to rapidly and effectively carry out a ligation reaction;

5) Library fragment screening

For the library added with the joint, a MagicPure DNA Beads II nucleic acid fragment screening kit is used for screening the size of the fragment while purifying the library; adopting two-step screening, firstly removing small fragments on the left side of a target region by SPRI magnetic beads, then removing large fragments on the right side of the target region, and finally screening out an original library with the fragment peak value of 250bp for the next PCR amplification; the library after purification removes excessive sequencing joints in the system and products of joint self-connection, avoids ineffective amplification in the PCR amplification process, and eliminates the influence on-machine sequencing;

6) PCR amplification of cDNA libraries

Amplifying the original library in a 50 μ L reaction system using high fidelity polymerase to ensure sufficient library inventory on the sequencer; only DNA fragments with joints on both ends can be amplified, and only the fragments connected with single-ended joints are removed, so that the qualified library is enriched; meanwhile, the newly generated double-strand cDNA is provided with dUTP, so that the amplification of high-fidelity polymerase using the dUTP as a template is prevented, the amplified library is only derived from the single-strand cDNA, and the strand direction information is faithfully reserved; the number of PCR amplification cycles is controlled between 12 and 15; on the premise of ensuring that the product is sufficient, the deviation caused by overlarge amplification cycle number is reduced; purifying the amplified library with magnetic beads to obtain a sequencing library which can be loaded on a computer;

7) Library quality detection

Performing quality inspection and quantification on the constructed sequencing library; accurately quantifying the concentration of the library by using the Qubit, determining the size distribution of the fragments of the library by using an Agilent 2100Bioanalyzer, and evaluating whether the library is suitable for being installed on a computer;

8) Sequencing on machine

Diluting samples qualified for quality inspection, and mixing and loading a plurality of samples according to corresponding molar proportions according to the sequencing flux requirements of different samples; sequencing the library by adopting an Illumina high-throughput sequencing platform and a 2 x 150bp double-end sequencing strategy;

(6) data analysis

1) mRNA raw data quality assessment

2) Statistics of sequencing data volume

3) Base mass distribution of raw data

4) Raw data filtering

5) Reference gene alignment

6) Quantitative expression of mRNA

7) Differential expression analysis of mRNA

8) Enrichment analysis of differentially expressed genes

9) Methylation site prediction

10 Analysis of methylation site differences

11 Methylation differential site Gene enrichment analysis

12 Combined analysis of methylation differential site genes with differentially expressed genes.

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