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.