Quantitative DNA-RNA Immunoprecipitation Sequencing with Spike-Ins.
Quantitative DNA-RNA Immunoprecipitation Sequencing with Spike-Ins.
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DOI：
10.1007/978-1-0716-2477-7_26
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发表时间：
2022
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R环是三链核酸结构，包括RNA-DNA杂合体和ssDNA的置换链。R环在细胞中具有重要的生理作用，但R环动力学的失调也会产生有害的细胞结果。R-loop的全基因组映射提供了一种在广泛背景下研究R-loop生物学的无偏见方法。在这里，我们提出了一个协议，测序RNA-DNA杂交全基因组链特异性和高分辨率。我们还提供了有关如何制备适当的内部加标标准品并将其纳入工作流程的信息，这些标准品有助于测序信号的准确标准化，从而为不同实验样品之间的R环形成提供定量见解。
R-loops are three-stranded nucleic acid structures, comprising an RNA-DNA hybrid and a displaced strand of ssDNA. R-loops have important physiological roles in cells, but deregulation of R-loop dynamics can also have harmful cellular outcomes. The genome-wide mapping of R-loops offers an unbiased approach to study R-loop biology in a wide-range of contexts. Here we present a protocol to sequence RNA-DNA hybrids genome-wide with strand-specificity and high resolution. We also include information on how to prepare and incorporate into the workflow appropriate internal spike-in standards which facilitate accurate normalization of the sequencing signal, thereby providing quantitative insights into R-loop formation between different experimental samples.