Open AccessQuantitative nucleotide resolution profiling of RNA cytidine acetylation by ac4C-seq
Author(s) -
Supuni Thalalla Gamage,
Aldema SasChen,
Schraga Schwartz,
Jordan L. Meier
Publication year - 2021
Publication title -
nature protocols
Language(s) - English
Resource type - Journals
eISSN - 1754-2189
pISSN - 1750-2799
DOI - 10.1038/s41596-021-00501-9
Subject(s) - rna , adapter (computing) , biology , computational biology , transcriptome , nucleotide , cytidine , rna seq , oligonucleotide , biochemistry , gene , gene expression , enzyme , electrical engineering , engineering
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.