Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, China.
Tsinghua-Peking Center for Life Sciences, Beijing, China.
Methods Mol Biol. 2022;2528:445-464. doi: 10.1007/978-1-0716-2477-7_29.
R-loop is a three-stranded chromatin structure, comprising one single-stranded DNA and another DNA:RNA hybrid strand, plays various and essential biological functions in many organisms. Developing a precise, efficient, faithful, and unbiased genome-wide R-loop detection method with extensive adaptability in all organisms is at the top priority for R-loop biology. Here, we provide a straightforward and highly efficient protocol for genome-wide strand-specific R-loop profiling in various organisms. In brief, genomic DNA is extracted and fragmented by the cocktail of restriction enzymes, and then the DNA:RNA hybrids are immunoprecipitated, following by the single-stranded DNA adaptor ligation and next-generation sequencing (named as ssDRIP-seq). Coupling with a straightforward and step-by-step bioinformatic pipeline, this method can provide high resolution and comprehensive strand-specific information for R-loop formation. ssDRIP-seq has been successfully applied for detecting R-loops from prokaryotes such as E. coli, to eukaryotes such as S. cerevisiae, mammalian cell culture and tissues, as well as plants Arabidopsis and rice, with high reproducibility and sensitivity.
R 环是一种三链染色质结构，由一条单链 DNA 和另一条 DNA:RNA 杂交链组成，在许多生物体中发挥着各种重要的生物学功能。开发一种精确、高效、忠实和无偏倚的全基因组 R 环检测方法，具有广泛的适应性，是 R 环生物学的首要任务。在这里，我们提供了一种在各种生物体中进行全基因组链特异性 R 环分析的简单而高效的方案。简而言之，提取基因组 DNA 并通过限制酶混合物进行片段化，然后免疫沉淀 DNA:RNA 杂交体，接着进行单链 DNA 接头连接和下一代测序（称为 ssDRIP-seq）。结合简单而逐步的生物信息学流程，该方法可以提供高分辨率和全面的链特异性 R 环形成信息。ssDRIP-seq 已成功应用于从原核生物如大肠杆菌，到真核生物如酿酒酵母、哺乳动物细胞培养物和组织，以及植物拟南芥和水稻中检测 R 环，具有高重复性和灵敏度。
