BID-seq for transcriptome-wide quantitative sequencing of mRNA pseudouridine at base resolution
Pseudouridine (?) is an abundant RNA modification that is present in and impacts the functions of diverse non-coding RNA species, including rRNA, tRNA, snRNA, etc. Pseudouridine also exists in mammalian mRNA and likely exhibits functional roles; however, functional investigations of pseudouridine in mammalian mRNA have been hampered by the lack of a quantitative method that detects ? at base precision. We have recently developed Bisulfite-Induced Deletion sequencing (BID-seq), which provides the community with a quantitative method to map RNA ? distribution transcriptome-wide at single-base resolution. Here, we describe an optimized BID-seq protocol to generate highly reproducible results, displaying nearly zero background deletions at unmodified uridines after bisulfite treatment and uncovering 8,407 ? sites starting from as little as 10 ng mouse embryonic stem cell (mESC) polyA+ RNA, with the steps that are fast in both library preparation and data analysis. 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. The 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 BID-seq, requiring only basic bioinformatic and computational skills to uncover ? signatures from NGS data. Overall design: [dataset1] 24 samples (Sample 1-24):  "Input" and "Treated" samples in BID-seq to detect pseudouridine in synthetic RNA oligos. [dataset2] 6 samples (Sample 25-30):  "Input" and "Treated" samples in BID-seq to detect pseudouridine in mESC polyA+ RNA.