4.8 Article

LEAD-m6A-seq for Locus-Specific Detection of N6-Methyladenosine and Quantification of Differential Methylation

期刊

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
卷 60, 期 2, 页码 873-880

出版社

WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.202007266

关键词

epitranscriptomics; N-6-methyladenosine; primer extension; RNA modification; RNA methylation

资金

  1. National Institute of Health [RM1 HG008935]
  2. MSTP training grant [T32GM007281]

向作者/读者索取更多资源

The study introduces a new method, LEAD-m(6)A-seq, which significantly improves the efficiency of locus-specific analysis of m(6)A modification by coupling it to next generation sequencing technology. This strategy allows for high-throughput validation and detection of m(6)A modification at selected sites, with high reproducibility in evaluating differential methylation levels.
N-6-methyladenosine (m(6)A) is a crucial RNA chemical mark which plays important roles in various biological processes. The development of highly multiplexed, cost-effective, and easy-to-operate methodologies for locus-specific analysis of m(6)A is critical for advancing our understanding of the roles of this modification. Herein, we report a method which builds upon the principle of the previously reported SELECT approach by significantly improving its efficiency and coupling it to next generation sequencing technology for high-throughput validation and detection of m(6)A modification at selected sites (LEAD-m(6)A-seq). Through probing cDNA extension mediated by Bst DNA polymerase at and near target cellular sites by sequencing, we evaluated m(6)A modification at these sites, and estimated differential methylation levels (0-84 %) upon in vitro demethylation by the m(6)A demethylase FTO with high reproducibility. We envision that this strategy can be readily used for testing a greater number of sites with a broad dynamic range and modified to study other RNA modifications.

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