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

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.

Automated summary

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.