Mild and ultrafast GLORI enables absolute quantification of m⁶A methylome from low-input samples

Methods for absolute quantification of N⁶-methyladenosine (m⁶A) have emerged as powerful tools in epitranscriptomics. We previously reported GLORI, a chemical-assisted approach to achieve unbiased and precise m⁶A measurement. However, its lengthy reaction time and severe RNA degradation have limited its applicability, particularly for low-input samples. Here, we present two updated GLORI approaches that are ultrafast, mild and enable absolute m⁶A quantification from one to two orders of magnitude less than the RNA starting material: GLORI 2.0 is compatible with RNA from ~10,000 cells and enhances sensitivity for both transcriptome-wide and locus-specific m⁶A detection; GLORI 3.0 further utilizes a reverse transcription-silent carrier RNA to achieve m⁶A quantification from as low as 500-1,000 cells. Using limited RNA from mouse dorsal hippocampus, we reveal a high modification level in synapse-related gene sets. We envision that the updated GLORI methods will greatly expand the applicability of absolute quantification of m⁶A in biology.