Single-base mapping of m6A by an antibody-independent method

N-6-methyladenosine (m(6)A) is one of the most abundant messenger RNA modifications in eukaryotes involved in various pivotal processes of RNA metabolism. The most popular high-throughput m(6)A identification method depends on the anti-m(6)A antibody but suffers from poor reproducibility and limited resolution. Exact location information is of great value for understanding the dynamics, machinery, and functions of m(6)A. Here, we developed a precise and high-throughput antibody-independent m(6)A identification method based on the m(6)A-sensitive RNA endoribonuclease recognizing ACA motif (m(6)A-sensitive RNA-Endoribonuclease-Facilitated sequencing or m(6)A-REF-seq). Whole-transcriptomic, single-base m(6)A maps generated by m(6)A-REF-seq quantitatively displayed an explicit distribution pattern with enrichment near stop codons. We used independent methods to validate methylation status and abundance of individual m(6)A sites, confirming the high reliability and accuracy of m(6)A-REF-seq. We applied this method on five tissues from human, mouse, and rat, showing that m(6)A sites are conserved with single-nucleotide specificity and tend to cluster among species.