Quantifying RNA Synthesis at Rate-Limiting Steps of Transcription Using Nascent RNA-Sequencing Data

Abstract

Nascent RNA-sequencing tracks transcription at nucleotide resolution. The genomic distribution of engaged transcription complexes, in turn, uncovers functional genomic regions. Here, we provide data-analytical steps to 1) identify transcribed regulatory elements de novo genome-wide, 2) quantify engaged transcription complexes at enhancers, promoter-proximal regions, divergent transcripts, gene bodies and termination windows, and 3) measure distribution of transcription machineries and regulatory proteins across functional genomic regions. This protocol follows RNA synthesis and genome-regulation in mammals, as demonstrated in human K562 erythroleukemia cells. For complete details on the use and execution of this protocol, please refer to Vihervaara, et al., 2021. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=128 HEIGHT=200 SRC="FIGDIR/small/454856v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@18301b2org.highwire.dtl.DTLVardef@1e4636forg.highwire.dtl.DTLVardef@13c1fd8org.highwire.dtl.DTLVardef@4eb935_HPS_FORMAT_FIGEXP M_FIG C_FIG