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

Methods that enable absolute quantification of N6-methyladenosine (m6A) RNA modification have emerged as powerful tools in the field of epitranscriptomics. We previously reported GLORI, a chemical-assisted approach firstly achieved quantitatively transcriptome-wide m6A measurement at single-base resolution. Despite its advantages, GLORI suffers from lengthy reaction time and severe RNA degradation. Here, we present two updated GLORI approaches: GLORI 2.0 is an ultra-fast and mild version that preserves RNA integrity and enhances sensitivity for both transcriptome-wide and locus-specific m6A detection; GLORI 3.0 further utilizes a novel reverse transcription-silent carrier RNA to achieve high-quality m6A quantification from ~ 1,000 cells. Using limited RNA input extracted from single mouse dorsal hippocampus, we measure m6A methylome in the synaptic and cytoplasmic fractions and 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. m6A profiling analysis of human cells and mice hippocampal using updated GLORI