RT Journal Article
SR Electronic
T1 Rapid and scalable profiling of nascent RNA with fastGRO
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.01.24.916015
DO 10.1101/2020.01.24.916015
A1 Elisa Barbieri
A1 Connor Hill
A1 Mathieu Quesnel-Vallieres
A1 Yoseph Barash
A1 Alessandro Gardini
YR 2020
UL http://biorxiv.org/content/early/2020/01/24/2020.01.24.916015.abstract
AB Genome-wide profiling of nascent RNA has become a fundamental tool to study transcription regulation. Over the past decade, next-generation sequencing has fostered development of a handful of techniques (i.e. GRO-seq, PRO-seq, TT-seq and NET-seq) that map unprocessed transcripts originating from both the coding and the noncoding portion of the genome. Unlike steady-state RNA sequencing, nascent RNA profiling mirrors the real-time activity of RNA Polymerases and provides an accurate readout of transcriptome-wide variations that occur during short time frames (i.e. response to external stimuli or rapid metabolic changes). Some species of nuclear RNAs, albeit functional, have a short half-life and can only be accurately gauged by nascent RNA techniques (i.e. lincRNAs and eRNAs). Furthermore, these techniques capture uncapped post-cleavage RNA at termination sites or promoter-associated antisense RNAs, providing a unique insight into RNAPII dynamics and processivity.Here we present a run-on assay with 4s-UTP labelling, followed by reversible biotinylation and affinity purification via streptavidin. Our protocol allows streamlined sample preparation within less than 3 days. We named the technique fastGRO (fast Global Run-On). We show that fastGRO is highly reproducible and yields a more complete and extensive coverage of nascent RNA than comparable techniques. Importantly, we demonstrate that fastGRO is scalable and can be performed with as few as 0.5×10^6 cells.