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

The N4-acetylcytidine RNA modification is conserved in all domains of life. Here, the authors provide a detailed protocol for whole-transcriptome quantitative nucleotide resolution mapping of this modified nucleobase in different organisms. 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.

Automated summary

The N4-acetylcytidine RNA modification is conserved in all domains of life. The authors present a detailed protocol for quantitative nucleotide resolution mapping of this modification, introducing N4-acetylcytidine sequencing (ac4C-seq) as a new method for accurate identification of specific ac4C residues and their level of modification. This protocol can be completed in approximately 4 days for transcriptome-wide sequencing.