GLORI for absolute quantification of transcriptome-wide mA at single-base resolution.

N-methyladenosine (mA) is the most abundant posttranscriptional chemical modification in mRNA, involved in regulating various physiological and pathological processes throughout mRNA metabolism. Recently, we developed GLORI, a sequencing method that enables the production of a globally absolute-quantitative mA map at single-base resolution. Our technique utilizes the glyoxal- and nitrite-based chemical reaction, which selectively deaminates unmethylated adenosines while leaving mA intact. The RNA library can then be prepared using a modified library construction protocol from enhanced UV crosslinking and immunoprecipitation (eCLIP) or commercial kits. Here we provide a detailed protocol for proper RNA sample handling and provide further guidelines for the use of a tailored bioinformatics pipeline (GLORI-tools) for subsequent data analysis. Compared with current methods, this new method is both exceptionally sensitive and robust, capable of identifying ~80,000 mA sites with 50 Gb sequencing data in mammalian cells. It also provides a quantitative readout for mA sites at single-base resolution. We hope the technique will provide a precise and unbiased tool for investigating mA biology across various fields. Basic expertise in molecular biology and knowledge of bioinformatics are required for the protocol. The entire procedure can be completed within a week, with the library construction process taking ~4 d.