Summary: As the most abundant internal mRNA modification, N6-methyladenosine (m6A) was involved in almost all the aspects of RNA metabolism. Here, we introduce our protocol for m6A-SAC-seq, which enables the whole transcriptome-wide mapping of m6A RNA modification at single-nucleotide resolution with stoichiometry information. m6A-SAC-seq relies on selective allyl labeling of m6A by specific methyltransferase and chemical treatment that introduce mutation upon reverse transcription. The technique only requires ∼30 ng of input RNA.For complete details on the use and execution of this protocol, please refer to Hu et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.