BID-seq for transcriptome-wide quantitative sequencing of mRNA pseudouridine at base resolution.
Li-Sheng ZhangChang YeCheng-Wei JuBoyang GaoXinran FengHui-Lung SunJiangbo WeiFan YangQing DaiChuan HePublished in: Nature protocols (2023)
Pseudouridine (Ψ) is an abundant RNA modification that is present in and affects the functions of diverse non-coding RNA species, including rRNA, tRNA and small nuclear RNA. Ψ also exists in mammalian mRNA and probably exhibits functional roles; however, functional investigations of mRNA Ψ modifications in mammals have been hampered by the lack of a quantitative method that detects Ψ at base precision. We have recently developed bisulfite-induced deletion sequencing (BID-seq), which provides the community with a quantitative method to map RNA Ψ distribution transcriptome-wide at single-base resolution. Here, we describe an optimized BID-seq protocol for mapping Ψ distribution across cellular mRNAs, which includes fast steps in both library preparation and data analysis. This protocol generates highly reproducible results by inducing high deletion ratios at Ψ modification within diverse sequence contexts, and meanwhile displayed almost zero background deletions at unmodified uridines. When used for transcriptome-wide Ψ profiling in mouse embryonic stem cells, the current protocol uncovered 8,407 Ψ sites from as little as 10 ng of polyA + RNA input. This optimized BID-seq workflow takes 5 days to complete and includes four main sections: RNA preparation, library construction, next-generation sequencing (NGS) and data analysis. Library construction can be completed by researchers who have basic knowledge and skills in molecular biology and genetics. In addition to the experimental protocol, we provide BID-pipe ( https://github.com/y9c/pseudoU-BIDseq ), a user-friendly data analysis pipeline for Ψ site detection and modification stoichiometry quantification, requiring only basic bioinformatic and computational skills to uncover Ψ signatures from BID-seq data.
Keyphrases
- data analysis
- single cell
- rna seq
- genome wide
- randomized controlled trial
- high resolution
- healthcare
- dna methylation
- embryonic stem cells
- nucleic acid
- gene expression
- single molecule
- copy number
- mass spectrometry
- mental health
- diabetic rats
- endothelial cells
- oxidative stress
- drug induced
- tandem mass spectrometry
- circulating tumor cells
- loop mediated isothermal amplification