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.

定义 RNA 修饰的转录组范围功能的先决条件是准确确定其位置的能力。在这里，我们提出了 N4-乙酰胞苷 (ac4C) 测序 (ac4C-seq)，这是一种用于 RNA 中胞苷乙酰化的定量单核苷酸分辨率映射的协议。该方法利用 ac4C 与氰基硼氢化钠在酸性条件下的动力学上容易的化学反应形成还原的核碱基。然后将 RNA 片段化，在其 3' 末端与接头连接并逆转录以在减少的 ac4C 位点引入非同源核苷酸。在接头连接、文库制备和高通量测序之后，生物信息学管道能够根据减少样本中存在的 C→T 错误掺入而不是对照中的存在来识别 ac4C 位置。与基于抗体的方法不同，ac4C-seq 可识别特定的 ac4C 残基并报告其修饰水平。ac4C-seq 文库制备协议可在约 4 d 内完成，用于转录组范围的测序。