Journal article
Quantitative nucleotide resolution profiling of RNA cytidine acetylation by ac4C-seq
Nature protocols, Vol.16, pp.2286-2307
Apr/2021
PMID: 33772246
Abstract
A prerequisite to defining the transcriptome-wide functions of RNA modifications is the ability to accurately determine their location. Here, we present N4-acetylcytidine (ac4C) sequencing (ac4C-seq), a protocol for the quantitative single-nucleotide resolution mapping of cytidine acetylation in RNA. This method exploits the kinetically facile chemical reaction of ac4C with sodium cyanoborohydride under acidic conditions to form a reduced nucleobase. RNA is then fragmented, ligated to an adapter at its 3' end and reverse transcribed to introduce a non-cognate nucleotide at reduced ac4C sites. After adapter ligation, library preparation and high-throughput sequencing, a bioinformatic pipeline enables identification of ac4C positions on the basis of the presence of C→T misincorporations in reduced samples but not in controls. Unlike antibody-based approaches, ac4C-seq identifies specific ac4C residues and reports on their level of modification. The ac4C-seq library preparation protocol can be completed in ~4 d for transcriptome-wide sequencing.
Details
- Title
- Quantitative nucleotide resolution profiling of RNA cytidine acetylation by ac4C-seq
- Creators
- Supuni Thalalla Gamage - National Cancer InstituteAldema Sas-Chen - 972WIS_INST___111Schraga Schwartz (Corresponding Author) - 972WIS_INST___111Jordan L Meier (Corresponding Author) - National Cancer Institute
- Resource Type
- Journal article
- Publication Details
- Nature protocols, Vol.16, pp.2286-2307; Apr/2021
- Number of pages
- 22
- Publisher
- England
- Language
- English
- DOI
- https://doi.org/10.1038/s41596-021-00501-9
- PMID
- 33772246
- Grant note
- We thank members of the Schwartz and Meier laboratories for many helpful comments. S.S. is funded by the Israel Science Foundation (543165), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 714023) and the Estate of Emile Mimran. S.S. is the incumbent of the Robert Edward and Roselyn Rich Manson Career Development Chair in Perpetuity. J.L.M. is supported by the Intramural Research Program of the National Institutes of Health (NIH), the National Cancer Institute, The Center for Cancer Research (ZIA BC011488-06). Author contribution - A.S.-C., S.T.G., J.L.M. and S.S. developed the protocol. A.S.-C. and S.T.G. performed the experiments. A.S.-C. designed the bioinformatic pipeline and analyzed the data. S.T.G. A.S.-C., S.S. and J.L.M. wrote the manuscript.
- Record Identifier
- 993265551303596
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