Deciphering the "mA Code" via Antibody-Independent Quantitative Profiling.

N6-methyladenosine (mA) is the most abundant modification on mRNA and is implicated in critical roles in development, physiology, and disease. A major limitation has been the inability to quantify mA stoichiometry and the lack of antibody-independent methodologies for interrogating mA. Here, we develop MAZTER-seq for systematic quantitative profiling of m6A at single-nucleotide resolution at 16%-25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of mA sites, calibration of the performance of antibody-based approaches, and quantitative tracking of mA dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is "hard coded" in cis via a simple and predictable code, accounting for 33%-46% of the variability in methylation levels and allowing accurate prediction of mA loss and acquisition events across evolution. MAZTER-seq allows quantitative investigation of mA regulation in subcellular fractions, diverse cell types, and disease states.