Enrichment of mRNA and Bisulfite-mRNA Library Preparation for Next-Generation Sequencing

Szu Ying Chen, Po Hsien Huang

研究成果: Article同行評審

摘要

RNA post-transcriptional modifications in various types of RNA transcripts are associated with diverse RNA regulation in eukaryotic cells. Aberrant RNA 5-methylcytosine modifications and the dysregulated expression of RNA methyltransferases have been shown to be associated with various diseases, including cancers. Transcriptome-wide bisulfite-sequencing was developed to characterize the positions and the quantitative cytosine methylation levels in the bisulfite-converted RNA at the base-pair resolution. Herein, this protocol presents the procedures of two rounds of poly(A) RNA purification, three cycles of bisulfite reaction, and library preparation in detail to allow the transcriptome-wide mapping of mRNA 5-methylcytosine modification sites. The assessment of RNA quantity and quality after the main reaction is essential to monitor RNA integrity and is a critical step for ensuring high-quality sequencing libraries. Ideally, the procedures can be completed within three days. With this protocol, using high-quality total RNA as the input can practically build up robust bisulfite-mRNA libraries for next-generation sequencing from the sample of interest.

原文English
文章編號e65352
期刊Journal of Visualized Experiments
2023
發行號197
DOIs
出版狀態Published - 2023 7月

All Science Journal Classification (ASJC) codes

  • 一般神經科學
  • 一般化學工程
  • 一般生物化學,遺傳學和分子生物學
  • 一般免疫學和微生物學

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