Pichot Florian, Marchand Virginie, Helm Mark, Motorin Yuri
Institute of Pharmacy of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Mainz, Germany.
Université de Lorraine, CNRS, INSERM, UAR2008/US40 IBSLor, EpiRNA-Seq Core facility, F-54000, Nancy, France.
Methods Mol Biol. 2023;2624:207-223. doi: 10.1007/978-1-0716-2962-8_14.
Pseudouridine, a modified RNA residue formed by the isomerization of its parental U nucleotide, is prevalent in a majority of cellular RNAs; its presence was reported in tRNA, rRNA, and sn/snoRNA as well as in mRNA/lncRNA. Multiple analytical deep sequencing-based approaches have been proposed for pseudouridine detection and quantification, among which the most popular relies on the use of soluble carbodiimide (termed CMCT). Recently, we developed an alternative protocol for pseudouridine mapping and quantification. The principle is based on protection of pseudouridine against random RNA cleavage by hydrazine/aniline treatment (HydraPsiSeq protocol). This "negative" detection mode requires higher sequencing depth and provides a precise quantification of the pseudouridine content. All "wet-lab" technical details of the HydraPsiSeq protocol have been described in recent publications. Here, we describe all bioinformatics analysis steps required for data processing from raw reads to the pseudouridylation profile of known or unknown RNA.
假尿苷是由其亲本尿苷酸异构化形成的一种修饰的RNA残基，在大多数细胞RNA中普遍存在；据报道，它存在于tRNA、rRNA、sn/snoRNA以及mRNA/lncRNA中。已经提出了多种基于深度测序分析的方法用于假尿苷的检测和定量，其中最常用的方法依赖于使用可溶性碳二亚胺（称为CMCT）。最近，我们开发了一种用于假尿苷定位和定量的替代方案。其原理基于通过肼/苯胺处理（HydraPsiSeq方案）保护假尿苷免受随机RNA切割。这种“阴性”检测模式需要更高的测序深度，并能对假尿苷含量进行精确量化。HydraPsiSeq方案的所有“湿实验室”技术细节已在最近的出版物中进行了描述。在这里，我们描述了从原始读数到已知或未知RNA的假尿苷化图谱的数据处理所需的所有生物信息学分析步骤。
