核糖核酸
适配器(计算)
计算生物学
转录组
RNA序列
核苷酸
胞苷
寡核苷酸
生物
生物化学
基因
基因表达
酶
电气工程
工程类
作者
Supuni Thalalla Gamage,Aldema Sas‐Chen,Schraga Schwartz,Jordan L. Meier
出处
期刊:Nature Protocols
[Springer Nature]
日期:2021-03-26
卷期号:16 (4): 2286-2307
被引量:57
标识
DOI:10.1038/s41596-021-00501-9
摘要
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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