Transcriptome-wide profiling and quantification of N 6 -methyladenosine by enzyme-assisted adenosine deamination

N 6 -methyladenosine (m 6 A), the most abundant internal messenger RNA modification in higher eukaryotes, serves myriad roles in regulating cellular processes. Functional dissection of m 6 A is, however, hampered in part by the lack of high-resolution and quantitative detection methods. Here we present evolved TadA-assisted N 6 -methyladenosine sequencing (eTAM-seq), an enzyme-assisted sequencing technology that detects and quantifies m 6 A by global adenosine deamination. With eTAM-seq, we analyze the transcriptome-wide distribution of m 6 A in HeLa and mouse embryonic stem cells. The enzymatic deamination route employed by eTAM-seq preserves RNA integrity, facilitating m 6 A detection from limited input samples. In addition to transcriptome-wide m 6 A profiling, we demonstrate site-specific, deep-sequencing-free m 6 A quantification with as few as ten cells, an input demand orders of magnitude lower than existing quantitative profiling methods. We envision that eTAM-seq will enable researchers to not only survey the m 6 A landscape at unprecedented resolution, but also detect m 6 A at user-specified loci with a simple workflow.