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.

N ⁶ -甲基腺苷(m ⁶ A)是mRNA中最丰富的转录后化学修饰，参与调节整个mRNA代谢的各种生理和病理过程。最近，我们开发了 GLORI，一种测序方法，能够以单碱基分辨率生成全局绝对定量 m ^{6 A 图谱。}我们的技术利用基于乙二醛和亚硝酸盐的化学反应，选择性地脱氨基未甲基化的腺苷，同时保持 m ⁶ A 完整。然后可以使用增强型 UV 交联和免疫沉淀 (eCLIP) 或商业试剂盒的修改文库构建方案来制备 RNA 文库。在这里，我们提供了正确处理 RNA 样本的详细协议，并提供了使用定制生物信息学管道（GLORI-工具）进行后续数据分析的进一步指南。与现有方法相比，这种新方法非常灵敏且稳健，能够利用哺乳动物细胞中的 50 Gb 测序数据识别约 80,000 m ^{6 A 位点。}它还以单碱基分辨率提供 m ^{6 A 位点的定量读数。}我们希望该技术能够为跨领域研究 m ⁶ A 生物学提供精确且公正的工具。该协议需要分子生物学的基本专业知识和生物信息学知识。整个过程可以在一周内完成，文库构建过程需要~4天。