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

Abstract 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.