Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination [eTAM-seq]

We report evolved TadA-assisted N6-methyladenosine sequencing (eTAM-seq), an enzyme-assisted sequencing technology for quantitative, base-resolution profiling of m6A. eTAM-seq functions by global adenosine deamination, enabling detection of m6A as persistent A. We demonstrate adenosine-to-inosine (I) conversion rates up to 99% using a hyperactive TadA variant. With eTAM-seq, we profile and quantify m6A in the whole transcriptomes of HeLa cells and mouse embryonic stem cells (mESCs), with simultaneous deconvolution of the transcriptome and epitranscriptome. Further, we showcase deep sequencing-free, site-specific m6A quantification with as few as 10 cells, an input demand that is at least 4 orders of magnitude lower than existing methods. Collectively, eTAM-seq enables sensitive detection and faithful quantification of m6A with limited RNA input, representing a novel solution to deciphering the epitranscriptome. Overall design: Nine HeLa wild type (3 FTO-, 3 FTO+ and 3 IVT), six mESC wild type (2 FTO-, 2 FTO+ and 2 IVT), two mESC Mettl3 cko ctrl (1 FTO- and 1 FTO+) and two mESC Mettl3 cko (1 FTO- and 1 FTO+) polyA samples were collected for high-throughput sequencing.