LEAD-m⁶ A-seq for Locus-Specific Detection of N⁶ -Methyladenosine and Quantification of Differential Methylation

N⁶ -methyladenosine (m⁶ 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 m⁶ 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 m⁶ A modification at selected sites (LEAD-m⁶ A-seq). Through probing cDNA extension mediated by Bst DNA polymerase at and near target cellular sites by sequencing, we evaluated m⁶ A modification at these sites, and estimated differential methylation levels (0-84 %) upon in vitro demethylation by the m⁶ 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.