N6-methyladenosine (m6A) 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 m6A map at single-base resolution. Our technique utilizes the glyoxal- and nitrite-based chemical reaction, which selectively deaminates unmethylated adenosines while leaving m6A 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 m6A sites with 50 Gb sequencing data in mammalian cells. It also provides a quantitative readout for m6A sites at single-base resolution. We hope the technique will provide a precise and unbiased tool for investigating m6A 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.
Key points: GLORI is a sequencing-based method for the characterization of N6-methyladenosine (m6A) methylome at single-base resolution. The procedure is based on the glyoxal- and nitrite-mediated deamination of unmethylated adenosine with high efficiency. A bioinformatic pipeline (GLORI-tool) is also complemented for optimized analysis of GLORI sequencing data.Compared with other m6A detection methods, GLORI demonstrates exceptional sensitivity and precision, providing a more comprehensive and clearer transcriptome-wide m6A stoichiometric landscape.
The formation and functional relevance of N6-methyladenosine sites are key unanswered questions in the field of RNA biology. The protocol describes an unbiased sequencing-based method for the characterization of the global distribution and stoichiometry of N6-methyladenosine sites.