Absolute quantification of single-base m6A methylation in the mammalian transcriptome using GLORI

N-6-methyladenosine (m(6)A) is the most abundant RNA modification in mammalian cells and the best-studied epitranscriptomic mark. Despite the development of various tools to map m(6)A, a transcriptome-wide method that enables absolute quantification of m(6)A at single-base resolution is lacking. Here we use glyoxal and nitrite-mediated deamination of unmethylated adenosines (GLORI) to develop an absolute m(6)A quantification method that is conceptually similar to bisulfite-sequencing-based quantification of DNA 5-methylcytosine. We apply GLORI to quantify the m(6)A methylomes of mouse and human cells and reveal clustered m(6)A modifications with differential distribution and stoichiometry. In addition, we characterize m(6)A dynamics under stress and examine the quantitative landscape of m(6)A modification in gene expression regulation. GLORI is an unbiased, convenient method for the absolute quantification of the m(6)A methylome. The m(6)A modification is mapped transcriptome-wide at single-base resolution in mammalian cells.