Abstract
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.
Key points
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This protocol describes a method for detecting and quantifying pseudouridine residues across the transcriptome.
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The protocol improves on previous pseudouridine sequencing methods by allowing base-resolution quantitative pseudouridine mapping while covering the entire transcriptome.
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Data availability
The underlying data of Fig. 2, Fig. 3 and Supplementary Fig. 1 have been deposited into the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo) under the accession number GSE238245. Source data are provided with this paper.
Code availability
Code for assessing the quality, reliability and features of the identified mRNA Ψ sites is available on GitHub (https://github.com/y9c/pseudoU-BIDseq).
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Acknowledgements
This project was supported by National Institutes of Health (NIH) grant RM1 HG008935 (to C.H.). The Mass Spectrometry Facility of the University of Chicago is funded by the National Science Foundation (CHE-1048528). We thank Pieter W. Faber and his team in the Genomics Facility of the University of Chicago for help with high-throughput sequencing.
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Authors and Affiliations
Contributions
C.H. and Q.D. supervised this project. L.-S.Z., C.-W.J. and C.Y. developed this optimized protocol. C.Y. developed the analysis pipeline (BID-pipe) and performed data analysis. C.-W.J. prepared the BID-seq libraries. B.G. and C.-W.J. performed stem cell culture and RNA purification. C.Y. and X.F. built data figures and supplementary tables. X.F., H.-L.S., J.W. and F.Y. provided general help on experiments. L.-S.Z., C.Y., C.-W.J., Q.D. and C.H. interpreted the results and wrote the manuscript.
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Competing interests
A patent application for BID-seq has been filed by The University of Chicago. C.H. is a scientific founder, member of the scientific advisory board and equity holder of Aferna Bio, Inc. and AccuaDX Inc.; a scientific cofounder and equity holder of Accent Therapeutics, Inc.; and a member of the scientific advisory board of Rona Therapeutics.
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Nature Protocols thanks Cynthia Burrows and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Related links
Key references using this protocol
Dai, Q. et al. Nat. Biotechnol. 41, 344–354 (2023): https://doi.org/10.1038/s41587-022-01505-w
Ge, R. et al. Nat. Protoc. 18, 626–657 (2023): https://doi.org/10.1038/s41596-022-00765-9
Hu, L. et al. Nat. Biotechnol. 40, 1210–1219 (2022): https://doi.org/10.1038/s41587-022-01243-z
Supplementary information
Supplementary Information
Supplementary Figs. 1–3
Supplementary Table 1 and 2
Supplementary Tables 1 and 2
Source data
Source Data Fig. 2
Statistical source data
Source Data Fig. 3
Statistical source data
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Zhang, LS., Ye, C., Ju, CW. et al. BID-seq for transcriptome-wide quantitative sequencing of mRNA pseudouridine at base resolution. Nat Protoc 19, 517–538 (2024). https://doi.org/10.1038/s41596-023-00917-5
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DOI: https://doi.org/10.1038/s41596-023-00917-5
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