Univ Lyon, École Normale Supérieure de Lyon, CNRS, UMR 5239, Laboratoire de Biologie et Modélisation de la Cellule, Lyon, France.
Methods Mol Biol. 2022;2528:411-428. doi: 10.1007/978-1-0716-2477-7_27.
By temporarily distorting the DNA double helix, the moving RNA polymerases can lead to the formation of non-B DNA structures. One of the most abundant and largest non-B DNA structures in the genome is the R-loop, a three-stranded structure forming when the nascent RNA hybridizes with its DNA template, thereby extruding the non-template DNA strand. Growing evidence suggests that at least a subset of R-loops could induce transcription stress and genome instability, although the direct, primary consequences of R-loop formation on the surrounding chromatin are still unclear.To understand the direct impact of R-loops on transcription and genome stability, accurate and quantitative mapping of R-loops is essential. R-loop mapping is commonly achieved using the antibody-based DNA:RNA Immunoprecipitation (DRIP) strategy. While it is reasonably straightforward to obtain robust DRIP enrichments from human cells, this has proved harder in yeast, where DRIP signals are often relatively weak, with a poor signal-to-noise ratio. Although it is unclear whether such weak signals stem from a technical or a biological reality, they make the accurate quantification of DRIP signals all the more important, especially when deep sequencing is used to monitor and quantify the distribution of R-loops genome-wide. Here we propose a DRIP protocol that has been optimized for the mapping and the quantification of R-loops in Schizosaccharomyces pombe but that can also be used in Saccharomyces cerevisiae. As a result, this protocol can be used to generate calibrated DRIP-seq data, where genomic DNA extracted from S. cerevisiae serves as spike-in reference.
通过暂时扭曲 DNA 双螺旋，移动的 RNA 聚合酶可以导致非 B DNA 结构的形成。基因组中最丰富和最大的非 B DNA 结构之一是 R 环，这是一种三链结构，当新生的 RNA 与它的 DNA 模板杂交时形成，从而将非模板 DNA 链挤出。越来越多的证据表明，至少一部分 R 环可能会引起转录应激和基因组不稳定性，尽管 R 环形成对周围染色质的直接、主要后果仍不清楚。为了了解 R 环对转录和基因组稳定性的直接影响，对 R 环进行准确和定量的映射是必不可少的。R 环的映射通常使用基于抗体的 DNA：RNA 免疫沉淀（DRIP）策略来实现。虽然从人类细胞中获得稳健的 DRIP 富集是相当简单的，但在酵母中，这证明更具挑战性，因为 DRIP 信号通常相对较弱，信噪比较差。虽然尚不清楚这些较弱的信号是源于技术还是生物学现实，但它们使得准确量化 DRIP 信号变得更加重要，尤其是当使用深度测序来监测和量化 R 环在全基因组范围内的分布时。在这里，我们提出了一种 DRIP 方案，该方案已针对酿酒酵母中的 R 环作图和定量进行了优化，但也可用于酿酒酵母。因此，该方案可用于生成校准的 DRIP-seq 数据，其中从酿酒酵母提取的基因组 DNA 用作 Spike-in 参考。
