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Quantitative nucleotide resolution profiling of RNA cytidine acetylation by ac4C-seq


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.

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Fig. 1: Overview of nucleobase reaction chemistry underlying ac4C-seq.
Fig. 2: Experimental workflow for ac4C-seq protocol.
Fig. 3: Flowchart illustrating steps involved in ac4C-seq.
Fig. 4: Quality control of RNA and cDNA libraries.
Fig. 5: Anticipated results of ac4C-seq.

Data availability

Results depicted in Fig. 5 are based on ac4C-seq data previously deposited in the Gene Expression Omnibus under accession number GSE135826 as part of the publication by Sas-Chen et al.5.

Code availability

The custom code used for the ‘Calculation of misincorporation level and statistical testing’ section is available at


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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).

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Authors and Affiliations



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.

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Correspondence to Schraga Schwartz or Jordan L. Meier.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

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Key references using this protocol

Sinclair, W. et al. ACS Chem. Biol. 12, 2922–2926 (2017):

Thomas, J. et al. J. Am. Chem. Soc. 140, 12667–12670 (2018):

Sas-Chen, A. et al. Nature 583, 638–643 (2020):

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Thalalla Gamage, S., Sas-Chen, A., Schwartz, S. et al. Quantitative nucleotide resolution profiling of RNA cytidine acetylation by ac4C-seq. Nat Protoc 16, 2286–2307 (2021).

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