Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University; Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University.
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University; Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University;
J Vis Exp. 2023 Jul 7(197). doi: 10.3791/65352.
RNA post-transcriptional modifications in various types of RNA transcripts are associated with diverse RNA regulation in eukaryotic cells. Aberrant RNA 5-methylcytosine modifications and the dysregulated expression of RNA methyltransferases have been shown to be associated with various diseases, including cancers. Transcriptome-wide bisulfite-sequencing was developed to characterize the positions and the quantitative cytosine methylation levels in the bisulfite-converted RNA at the base-pair resolution. Herein, this protocol presents the procedures of two rounds of poly(A) RNA purification, three cycles of bisulfite reaction, and library preparation in detail to allow the transcriptome-wide mapping of mRNA 5-methylcytosine modification sites. The assessment of RNA quantity and quality after the main reaction is essential to monitor RNA integrity and is a critical step for ensuring high-quality sequencing libraries. Ideally, the procedures can be completed within three days. With this protocol, using high-quality total RNA as the input can practically build up robust bisulfite-mRNA libraries for next-generation sequencing from the sample of interest.
各种类型的 RNA 转录本中的 RNA 转录后修饰与真核细胞中多样化的 RNA 调控有关。异常的 RNA 5-甲基胞嘧啶修饰和 RNA 甲基转移酶的失调表达已被证明与各种疾病有关，包括癌症。全转录组亚硫酸氢盐测序技术的发展，能够以碱基分辨率对经亚硫酸氢盐转化的 RNA 中的位置和定量胞嘧啶甲基化水平进行特征描述。本文详细介绍了两轮 poly(A) RNA 纯化、三轮亚硫酸氢盐反应和文库制备的过程，以实现 mRNA 5-甲基胞嘧啶修饰位点的全转录组图谱绘制。主反应后评估 RNA 的数量和质量对于监测 RNA 的完整性至关重要，也是确保高质量测序文库的关键步骤。理想情况下，这些步骤可以在三天内完成。使用本方案，以高质量的总 RNA 作为输入，可以从感兴趣的样本中实际构建用于下一代测序的稳健的亚硫酸氢盐-mRNA 文库。
