Targeted Nuclease Approaches for Mapping Native R-Loops.
Targeted Nuclease Approaches for Mapping Native R-Loops.
复制标题
用于绘制天然 R 环的靶向核酸酶方法。
DOI：
10.1007/978-1-0716-2477-7_25
复制
发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Sarma,Kavitha
中科院分区：
文献类型：
--
作者：
Wulfridge,Phillip;Yan,Qingqing;Sarma,Kavitha
R 环是三链 DNA:RNA 杂合结构，由于转录而在整个基因组中自然形成。准确确定 R 环的基因组位置和链起源对于了解它们在基因调控和疾病中的作用至关重要。在这里，我们描述了一种基于核酸酶的全基因组和链特异性 R 环检测协议，我们将其称为 MapR。该方法使用修饰的 RNase H 酶以天然 R 环为目标进行切割和释放，然后进行深度测序。该协议的扩展 BisMapR 还可以通过 R 环单链 DNA 成分的非变性亚硫酸氢盐转化来引入链特异性。 MapR 和 BisMapR 识别具有高分辨率和低背景的 R 环，可以在低细胞输入下进行，并且需要较短的实验时间。
R-loops are three-stranded, DNA:RNA hybrid-containing structures that form naturally throughout the genome as a consequence of transcription. Accurately determining the genomic locations and strand of origin of R-loops is critical to understanding their roles in gene regulation and disease. Here, we describe a nuclease-based protocol for genome-wide and strand-specific R-loop detection, which we term MapR. This method targets native R-loops for cleavage and release using a modified RNase H enzyme, followed by deep sequencing. An extension of the protocol, BisMapR, can additionally introduce strand specificity via non-denaturing bisulfite conversion of the R-loop’s single-stranded DNA component. MapR and BisMapR identify R-loops with high resolution and low background, can be performed with low cell input, and require short experimental time.