Single-base mapping of m 6 A 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.