Use of the nuclear walk-on methodology to determine sites of RNA polymerase II initiation and pausing and quantify nascent RNAs in cells.

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.