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GLORI for absolute quantification of transcriptome-wide m6A at single-base resolution

文献类型： 外文期刊

作者： Shen, Weiguo 1 ; Sun, Hanxiao 2 ; Liu, Cong 2 ; Yi, Yunpeng 1 ; Hou, Yongkang 1 ; Xiao, Ye 4 ; Hu, Yufei 1 ; Lu, Bo 2 ; Peng, Jinying 2 ; Wang, Jing 1 ; Yi, Chengqi 2 ;

作者机构： 1.Peking Univ, Sch Pharmaceut Sci, Dept Chem Biol, State Key Lab Nat & Biomimet Drugs, Beijing, Peoples R China

2.Peking Univ, Sch Life Sci, State Key Lab Prot & Plant Gene Res, Beijing, Peoples R China

3.Shandong Acad Agr Sci, Inst Poultry Sci, Shandong Prov Anim & Poultry Green Hlth Prod Creat, Jinan, Peoples R China

4.Peking Univ, Peking Tsinghua Ctr Life Sci, Beijing, Peoples R China

5.Peking Univ, Coll Chem & Mol Engn, Dept Chem Biol, Beijing, Peoples R China

6.Peking Univ, Coll Chem & Mol Engn, Synthet & Funct Biomol Ctr, Beijing, Peoples R China

期刊名称：NATURE PROTOCOLS （ 影响因子：14.8； 五年影响因子：16.9 ）

ISSN： 1754-2189

年卷期： 2024 年 19 卷 4 期

页码：

收录情况： SCI

摘要： N-6-methyladenosine (m(6)A) 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 m(6)A map at single-base resolution. Our technique utilizes the glyoxal- and nitrite-based chemical reaction, which selectively deaminates unmethylated adenosines while leaving m(6)A 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 similar to 80,000 m(6)A sites with 50 Gb sequencing data in mammalian cells. It also provides a quantitative readout for m(6)A sites at single-base resolution. We hope the technique will provide a precise and unbiased tool for investigating m(6)A 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 similar to 4 d.

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