Library construction method and application of rna 5mC bisulfite sequencing

A bisulfite, sequencing library technology, applied in the application field of 5mC high-throughput sequencing, can solve problems such as the assembly form of transcripts affecting the retention level of exons, improve the efficiency of reverse transcription synthesis, and achieve comprehensive and complete detection. , the effect of improving efficiency

Active Publication Date: 2018-02-06
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

Based on existing studies, it is speculated that the modification of 5mC may regulate the alternative splicing of mRNA, and the level of 5mC modification may affect the retention level of exons and the assembly form of transcripts

Method used

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  • Library construction method and application of rna 5mC bisulfite sequencing
  • Library construction method and application of rna 5mC bisulfite sequencing
  • Library construction method and application of rna 5mC bisulfite sequencing

Examples

Experimental program
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Effect test

Embodiment 1

[0083] 1) RNA extraction and fragmentation

[0084] Using the T7 in vitro transcription kit (NEB, E2040S) to transcribe the RNA sequence of DHFR from the plasmid pcDNA3-HA-mDHFR carrying the full length of the mouse DHFR gene, and use it as a standard for identifying the conversion efficiency of bisulfite. Total RNA was extracted from human HeLa cells by Trizol method. according to The mRNA Purification Kit (Ambion, 61006) explains the isolation and purification of mRNA from total RNA. Mix 2 μg mRNA and 20 ng DHFR-RNA standard obtained by in vitro transcription to form an mRNA sample, and fragment the mRNA sample into 100 nt fragments according to the method instructions of the Ambion RNA Fragmentation Kit. The fragmentation condition was 2 μg mRNA / 9 μl for each reaction, 1 μl of fragmentation reagent was added, reacted at 90° C. for 1 minute, and then 1 μl of stop buffer was added. Add glycogen, 3M sodium acetate (pH 5.2) and pre-cooled pure ethanol to the reacted mRNA pr...

Embodiment 2

[0099] Download the human genome annotation file (version number 72) from the Ensembl database. The methylation sites obtained by sequencing analysis were annotated with Bedtools software based on the downloaded annotation files. The genes containing 5mC modification in the annotation results were classified based on the classification of genes in ensembl. The results showed that 87% of the genes containing 5mC methylation belonged to protein-coding genes ( Figure 4 A), the remaining modified genes are non-coding genes, including pseudogene, lincRNA, antisense, processed transcript, etc. Among them, there are 202 5mC modified pseudogenes, 62 lincRNAs, 58 antisenses, and 39 processed transcripts ( Figure 4 B).

Embodiment 3

[0101] In order to examine the distribution of 5mC modifications in various regions of the transcript, the transcript was first divided into four regions according to the annotation information of the Ensembl database, 5' non-coding region (5'UTR), protein coding region (CDS), internal Intron (Intron) and 3' non-coding region (3'UTR). The positional information of the 5mC modification site on the mRNA in Example 1 was extracted, analyzed by Bedtools software and the annotation information downloaded from the Ensembl database, and detected that these methylation sites were in four regions of the mRNA (5'UTR, CDS, Intron and 3'UTR), the results showed that the distribution of these methylation sites in CDS was 35%, in introns was 28%, in 3' non-coding regions was 24%, and in 5' non-coding regions was 13% ( Figure 5 A). Calculate the distribution ratio of the C sites (coverage times greater than or equal to 1) on the mRNA measured in the sequencing results in the four regions ...

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Abstract

The invention relates to a library construction method, a sequencing method and a methylation detection method for sequencing 5-methylcytosine (5-methylcytosine, 5mC) modified bisulfite on RNA molecules. By designing and using a three-base random hexamer primer containing only ACT, the efficiency of reverse transcription and PCR amplification of RNA fragments treated with bisulfite and the detection efficiency of 5mC sites are significantly improved, which is beneficial to the detection of 5mC sites high-throughput sequencing.

Description

technical field [0001] The invention belongs to the technical field of genome sequencing, and in particular relates to a library construction method after reverse transcription of bisulfite-treated RNA fragments by using random hexamer primers containing only ACT three bases and its 5mC Qualcomm applications in quantitative sequencing. Background technique [0002] RNA has dual functions as a regulatory and information molecule, and plays a central role in many cellular mechanisms. Post-transcriptional modifications of RNA provide the chemical basis for the diversification of RNA functions. RNA modifications in nature widely exist on four types of nucleotides, A, U, C, and G. Up to now, the RNA modification database RNAMDB has included a total of 109 RNA modification forms, among which methylation modification accounts for about 80% of the total RNA modification, and this type of methylation modification mainly occurs on the nitrogen atom N of the base group , and on the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/6806C12Q1/6869C40B50/06
Inventor 杨运桂杨莹孙宝发杨鑫孙慧颖孙敏张兵黄春敏
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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