GSE201063
Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination [site_specific_quantification]
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: Two gene (ACTB and EIF2A) transcripts from serial diluted samples. Two gene (ACTB and EIF2A) transcripts from limited total RNA samples. 16 gene transcripts with in vio or in vitro transcription. Four gene transcripts with in vio transcription.