Abstract
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
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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.
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Compared with other m6A detection methods, GLORI demonstrates exceptional sensitivity and precision, providing a more comprehensive and clearer transcriptome-wide m6A stoichiometric landscape.
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Data availability
The sequence data generated in this study have been deposited in the NCBI Gene Expression Omnibus, under accession code GSE233875.
Code availability
The code of GLORI-tools is freely available on GitHub (https://github.com/liucongcas/GLORI-tools).
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Acknowledgements
The authors thank the State Key Laboratory of Natural and Biomimetic Drugs at Peking University for their advice and technological support, the High-Performance Computing Platform of the Center for Life Science for their analysis assistance and the National Center for Protein Sciences at Peking University in Beijing, China for their aid in quantifying the library size distribution. This work was supported by the National Natural Science Foundation of China (nos. 22077006 to J.W., 91940304 to C.Y., 91853107 to J.W. and 21825701 to C.Y.) and the National Key R&D Program of China (nos. 2022YFC3400600 to J.W., 2019YFA0802201 to C.Y., 2017YFA0505202 to J.W., 2019YFA0110902 to C.Y. and 2020YFA0710401 to J.P.).
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The project was conceived and the experiments were designed by J.W. and C.Y. The manuscript was written and edited by C.Y., J.W. and W.S. with the help of C.L., H.S., Y. Hou. and Y. Hu. The chemical method of GLORI was developed by J.W. and Y.Y. Experiments were performed by W.S. and H.S., and sample preparation for next-generation sequencing (NGS) was designed and conducted by H.S. with the help of Y.X. and B.L. The bioinformatics pipeline of GLORI was developed by C.L., who also performed data analysis with the assistance of C.Y. Further optimization of the reaction condition was carried out by W.S. and H.S. The project was supervised by C.Y. and J.W.
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Key reference using this protocol
Liu, C. et al. Nat Biotechnol. 41, 355–366 (2023): https://doi.org/10.1038/s41587-022-01487-9
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Supplementary Information
Supplementary Figs. 1 and 2.
Supplementary Tables 1–3
Sequences and primers.
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Shen, W., Sun, H., Liu, C. et al. GLORI for absolute quantification of transcriptome-wide m6A at single-base resolution. Nat Protoc 19, 1252–1287 (2024). https://doi.org/10.1038/s41596-023-00937-1
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DOI: https://doi.org/10.1038/s41596-023-00937-1
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