Use of the Nuclear Walk-on Methodology to Determine Sites of RNA Polymerase II Initiation and Pausing and Quantify Nascent RNAs in Cells,Methods

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Use of the Nuclear Walk-on Methodology to Determine Sites of RNA Polymerase II Initiation and Pausing and Quantify Nascent RNAs in Cells
Methods ( IF 4.2 ) Pub Date : 2019-04-01 , DOI: 10.1016/j.ymeth.2019.02.003
Christopher B Ball ₁ , Kyle A Nilson ₂ , David H Price ₁

Affiliation

Transcription by RNA polymerase II (Pol II) is controlled during initiation, elongation, and termination by a large variety of transcription factors, the state of chromatin modifications, and environmental conditions. Herein we describe experimental approaches for the examination of Pol II transcription at semi-global and genome-wide scales through analysis of nascent Pol II transcripts. We begin with a description of the nuclear walk-on (NWO) assay, which involves rapid isolation of nuclei in the presence of EDTA, followed by extension of about a quarter of the nascent transcripts with 32P-CTP. Labeled nascent transcripts are then analyzed by denaturing PAGE and phosphorimaging followed by densitometry analysis to quantify the signal on the gel. A parallel reaction containing α-amanitin to inhibit Pol II reveals transcription due to Pol I and Pol III, which can be subtracted to yield a profile of Pol II transcription. We then describe how to use the NWO as a front end for PRO-Seq and PRO-Cap methods, which permit the genome-wide characterization of Pol II transcription at nucleotide resolution and provide precise information about sites of transcription initiation and pausing. We discuss strategies for optimizing sequencing methods that capture nascent Pol II transcripts, methods of bias reduction, and approaches for normalizing these and other sequencing datasets using spike-in controls.

中文翻译：

使用核行走方法确定 RNA 聚合酶 II 起始和暂停位点并定量细胞中的新生 RNA

RNA 聚合酶 II (Pol II) 的转录在起始、延伸和终止过程中受到多种转录因子、染色质修饰状态和环境条件的控制。在此，我们描述了通过分析新生 Pol II 转录本在半全局和全基因组范围内检查 Pol II 转录的实验方法。我们首先描述核行走 (NWO) 测定，该测定涉及在 EDTA 存在下快速分离细胞核，然后用 32P-CTP 延伸约四分之一的新生转录本。然后通过变性 PAGE 和磷光成像对标记的新生转录本进行分析，然后进行光密度分析以量化凝胶上的信号。含有 α-鹅膏蕈碱抑制 Pol II 的平行反应揭示了由 Pol I 和 Pol III 引起的转录，可以将其减去以产生 Pol II 转录的概况。然后，我们描述了如何使用 NWO 作为 PRO-Seq 和 PRO-Cap 方法的前端，这些方法允许以核苷酸分辨率对 Pol II 转录进行全基因组表征，并提供有关转录起始和暂停位点的精确信息。我们讨论了优化捕获新生 Pol II 转录本的测序方法的策略、减少偏差的方法，以及使用掺入对照对这些和其他测序数据集进行标准化的方法。

更新日期：2019-04-01

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