Quantitative and whole-transcriptome N6-Methyladenosine profiling at single-nucleotide resolution [polyA_RNA]

We introduce m6A Selective Allyl Chemical labeling and Sequencing (m6A-SAC-Seq), a novel method for transcriptome-wide quantitative mapping of m6A at single-nucleotide resolution. The m6A-SAC-Seq employs a dimethyltransferase to selectively label m6A followed by introducing mutations with reverse transcriptase during sequencing. We identified the widespread distributions of m6A and quantitated their fractions in the transcriptome of HeLa, HEK293, HepG2, and human CD34+ hematopoietic stem/progenitor cells (HSPCs). Overall design: The RNA is treated with recombinant MjDim1 enzyme with allylic-SAM cofactor under optimized conditions to selectively convert m6A residues to a6m6A, followed by adding I2 to convert am6A to EA. Reverse transcription induces mutations at EA sites, enabling identification of the sites labeled with a6m6A.