Abstract:

Quantifying crucial steps in gene regulation during transcription elongation, such as promoter-proximal pausing, requires high resolution methods to map the transcription machinery across the genome. Native Elongating Transcript sequencing (NET-seq) interrogates the 3' ends of nascent RNA through sequencing, providing a direct visualization of RNA Polymerase II (Pol II) positions genome-wide with strand specificity and single nucleotide resolution. NET-seq applied to human cells has uncovered regions of Pol II pausing at the boundaries of retained exons and convergent antisense transcription near transcription start sites (Mayer et al. 2015). It has also been used to investigate regulators of productive elongation (Winter et al. 2017), and the directionality of promoter regions (Jin et al. 2017). Here, we describe the experimental protocol for metazoan cells that includes a spike-in control enabling normalization across samples. We also report on an improved bioinformatics pipeline for NET-seq. Together, the protocol yields a fast and non-perturbative method to map Pol II transcription genome-wide, revealing complex and global transcriptional events.