Department of Molecular and Cellular Biology and Genome Center, University of California, Davis, Davis, CA, USA.
Nat Protoc. 2019 Jun;14(6):1734-1755. doi: 10.1038/s41596-019-0159-1. Epub 2019 May 3.
R-loops are prevalent three-stranded non-B DNA structures composed of an RNA-DNA hybrid and a single strand of DNA. R-loops are implicated in various basic nuclear processes, such as class-switch recombination, transcription termination and chromatin patterning. Perturbations in R-loop metabolism have been linked to genomic instability and have been implicated in human disorders, including cancer. As a consequence, the accurate mapping of these structures has been of increasing interest in recent years. Here, we describe two related immunoprecipitation-based methods for mapping R-loop structures: basic DRIP-seq (DNA-RNA immunoprecipitation followed by high-throughput DNA sequencing), an easy, robust, but resolution-limited technique; and DRIPc-seq (DNA-RNA immunoprecipitation followed by cDNA conversion coupled to high-throughput sequencing), a high-resolution and strand-specific iteration of the method that permits accurate R-loop mapping genome wide. Briefly, after gentle DNA extraction and restriction digestion with a cocktail of enzymes, R-loop structures are immunoprecipitated with the anti-RNA-DNA hybrid S9.6 antibody. Compared with DRIP-seq, in which the immunoprecipitated DNA is directly sequenced, DRIPc-seq permits the recovery of the RNA moiety of R-loops, and these RNA strands are subjected to strand-specific RNA sequencing (RNA-seq) analysis. DRIPc-seq can be performed in 5 d and can be applied to any cell type, provided sufficient starting material can be collected. Accurately mapping R-loop distribution in various cell lines and under varied conditions is essential to understanding the formation, roles and dynamic resolution of these important structures.
R 环是由 RNA-DNA 杂交体和单链 DNA 组成的三链非 B-DNA 结构，普遍存在。R 环参与多种基本核过程，如类别转换重组、转录终止和染色质模式形成。R 环代谢的扰动与基因组不稳定性有关，并与包括癌症在内的人类疾病有关。因此，近年来，这些结构的准确作图越来越受到关注。在这里，我们描述了两种基于免疫沉淀的 R 环结构作图方法：基本 DRIP-seq（DNA-RNA 免疫沉淀 followed by high-throughput DNA sequencing），一种简单、稳健但分辨率有限的技术；以及 DRIPc-seq（DNA-RNA 免疫沉淀 followed by cDNA 转换 coupled to high-throughput sequencing），该方法是一种分辨率更高且具有链特异性的迭代方法，可在全基因组范围内进行准确的 R 环作图。简而言之，在温和的 DNA 提取和用酶混合物进行限制消化后，用抗 RNA-DNA 杂交体 S9.6 抗体进行 R 环结构免疫沉淀。与 DRIP-seq 相比，在 DRIP-seq 中，直接对免疫沉淀的 DNA 进行测序，而 DRIPc-seq 允许回收 R 环的 RNA 部分，并且这些 RNA 链进行链特异性 RNA 测序（RNA-seq）分析。DRIPc-seq 可以在 5 天内完成，并且可以应用于任何细胞类型，只要可以收集到足够的起始材料。准确绘制各种细胞系和不同条件下 R 环的分布对于理解这些重要结构的形成、作用和动态分辨率至关重要。
