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

N⁶-methyladenosine (m⁶A) is the most abundant RNA modification in mammalian cells and the best-studied epitranscriptomic mark. Despite the development of various tools to map m⁶A, a transcriptome-wide method that enables absolute quantification of m⁶A at single-base resolution is lacking. Here we use glyoxal and nitrite-mediated deamination of unmethylated adenosines (GLORI) to develop an absolute m⁶A quantification method that is conceptually similar to bisulfite-sequencing-based quantification of DNA 5-methylcytosine. We apply GLORI to quantify the m⁶A methylomes of mouse and human cells and reveal clustered m⁶A modifications with differential distribution and stoichiometry. In addition, we characterize m⁶A dynamics under stress and examine the quantitative landscape of m⁶A modification in gene expression regulation. GLORI is an unbiased, convenient method for the absolute quantification of the m⁶A methylome.