Université de Lorraine, CNRS, INSERM, UMS2008/US40 IBSLor, EpiRNA-Seq Core facility, F-54000 Nancy, France.
Université de Lorraine, CNRS, INSERM, UMS2008/US40 IBSLor, EpiRNA-Seq Core facility, F-54000 Nancy, France; Université de Lorraine, CNRS, UMR7365 IMoPA, F-54000 Nancy, France.
Methods. 2022 Jul;203:383-391. doi: 10.1016/j.ymeth.2021.08.008. Epub 2021 Sep 1.
Detection of RNA modified nucleotides using deep sequencing can be performed by several approaches, including antibody-driven enrichment and natural or chemically induced RT signatures. However, only very few RNA modified nucleotides generate natural RT signatures and antibody-driven enrichment heavily depends on the quality of antibodies used and may be highly biased. Thus, the use of chemically-induced RT signatures is now considered as the most trusted experimental approach. In addition, the use of chemical reagents allows inclusion of simple "mock-treated" controls, to exclude spontaneous RT arrests, SNPs and other misincorporation-prone sites. Hydrazine is a well-known RNA-specific reagent, already extensively used in the past for RNA sequencing and structural probing. Hydrazine is highly reactive to U and shows low reaction rates with ψ residues, allowing their distinction by deep sequencing-based protocols. However, other modified RNA residues also show particular behavior upon hydrazine treatment. Here we present methodological developments allowing to use HydraPsiSeq for precise quantification of RNA pseudouridylation and also detection and quantification of some other RNA modifications, in addition to ψ residues.
使用深度测序检测 RNA 修饰核苷酸可以通过几种方法来实现，包括抗体驱动的富集和天然或化学诱导的 RT 特征。然而，只有极少数的 RNA 修饰核苷酸会产生天然的 RT 特征，而抗体驱动的富集严重依赖于所用抗体的质量，并且可能存在很大的偏向性。因此，现在使用化学诱导的 RT 特征被认为是最可靠的实验方法。此外，化学试剂的使用允许包括简单的“模拟处理”对照，以排除自发的 RT 停顿、单核苷酸多态性和其他易发生错误掺入的位点。水合肼是一种众所周知的 RNA 特异性试剂，过去已广泛用于 RNA 测序和结构探测。水合肼与 U 高度反应，与 ψ 残基的反应速率较低，允许通过基于深度测序的方案来区分它们。然而，其他修饰的 RNA 残基在水合肼处理后也表现出特殊的行为。在这里，我们提出了一些方法学上的发展，允许使用 HydraPsiSeq 来精确定量 RNA 假尿嘧啶化，以及检测和定量除 ψ 残基以外的一些其他 RNA 修饰。
