Pan Yi, Adachi Hironori, He Xueyang, Chen Jonathan L, Yu Yi-Tao, Boutz Paul L
University of Rochester Medical Center, Department of Biochemistry and Biophysics, Center for RNA Biology, Rochester, NY, USA.
Bio Protoc. 2024 May 5;14(9):e4985. doi: 10.21769/BioProtoc.4985.
Pseudouridine (Ψ), the most prevalent modified base in cellular RNAs, has been mapped to numerous sites not only in rRNAs, tRNAs, and snRNAs but also mRNAs. Although there have been multiple techniques to identify Ψs, due to the recent development of sequencing technologies some reagents are not compatible with the current sequencer. Here, we show the updated Pseudo-seq, a technique enabling the genome-wide identification of pseudouridylation sites with single-nucleotide precision. We provide a comprehensive description of Pseudo-seq, covering protocols for RNA isolation from human cells, library preparation, and detailed data analysis procedures. The methodology presented is easily adaptable to any cell or tissue type with high-quality mRNA isolation. It can be used for discovering novel pseudouridylation sites, thus constituting a crucial initial step toward understanding the regulation and function of this modification. Key features • Identification of Ψ sites on mRNAs. • Updated Pseudo-seq provides precise positional and quantitative information of Ψ. • Uses a more efficient library preparation with the latest, currently available materials.
假尿苷（Ψ）是细胞RNA中最普遍的修饰碱基，不仅已被定位到rRNA、tRNA和snRNA中的众多位点，还存在于mRNA中。尽管已有多种技术用于鉴定Ψ，但由于测序技术的最新发展，一些试剂与当前的测序仪不兼容。在此，我们展示了更新后的假尿苷测序（Pseudo-seq）技术，这是一种能够以单核苷酸精度在全基因组范围内鉴定假尿苷化位点的技术。我们对Pseudo-seq进行了全面描述，涵盖了从人类细胞中分离RNA、文库制备以及详细的数据分析程序等方案。所介绍的方法很容易适用于任何能够高质量分离mRNA的细胞或组织类型。它可用于发现新的假尿苷化位点，从而构成理解这种修饰的调控和功能的关键第一步。关键特性 • 鉴定mRNA上的Ψ位点。 • 更新后的Pseudo-seq提供Ψ的精确位置和定量信息。 • 使用最新的可用材料进行更高效的文库制备。
