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). Display omitted •A protocol to label RNA m6A with allyl group•Incorporate mismatch and detect m6A at single-nucleotide resolution•Provide stoichiometric information of individual m6A sites with spike-in calibration•An optimized assay to detect and quantitate RNA m6A with only ∼ 30 ng input RNA Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.