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

Supuni Thalalla Gamage, Aldema Sas-Chen, Schraga Schwartz*, Jordan L. Meier*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Pages (from-to)2286-2307
Number of pages22
JournalNature Protocols
Issue number4
StatePublished - Apr 2021
Externally publishedYes


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