Quantitative nucleotide resolution profiling of RNA cytidine acetylation by ac4C-seq.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
04 2021
04 2021
Historique:
received:
23
08
2020
accepted:
13
01
2021
pubmed:
28
3
2021
medline:
5
5
2021
entrez:
27
3
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 33772246
doi: 10.1038/s41596-021-00501-9
pii: 10.1038/s41596-021-00501-9
pmc: PMC9103714
mid: NIHMS1800118
doi:
Substances chimiques
Nucleotides
0
Cytidine
5CSZ8459RP
RNA
63231-63-0
Types de publication
Journal Article
Research Support, N.I.H., Intramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
2286-2307Subventions
Organisme : Intramural NIH HHS
ID : ZIA BC011488
Pays : United States
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