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Rapid and scalable profiling of nascent RNA with fastGRO

Elisa Barbieri, Connor Hill, Mathieu Quesnel-Vallieres, Yoseph Barash, Alessandro Gardini
doi: https://doi.org/10.1101/2020.01.24.916015
Elisa Barbieri
1The Wistar Institute, Gene Expression and Regulation Program, 3601 Spruce street, 19104, Philadelphia, PA, U.S
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Connor Hill
1The Wistar Institute, Gene Expression and Regulation Program, 3601 Spruce street, 19104, Philadelphia, PA, U.S
2Cell and Molecular Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, 3400 Civic Center Boulevard, 19104, Philadelphia, PA, U.S
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Mathieu Quesnel-Vallieres
3Department of Genetics, Perelman School of Medicine, University of Pennsylvania, 3400 Civic Center Boulevard, 19104, Philadelphia, PA, U.S
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Yoseph Barash
3Department of Genetics, Perelman School of Medicine, University of Pennsylvania, 3400 Civic Center Boulevard, 19104, Philadelphia, PA, U.S
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Alessandro Gardini
1The Wistar Institute, Gene Expression and Regulation Program, 3601 Spruce street, 19104, Philadelphia, PA, U.S
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  • For correspondence: agardini@wistar.org
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Abstract

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.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted January 24, 2020.
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Rapid and scalable profiling of nascent RNA with fastGRO
Elisa Barbieri, Connor Hill, Mathieu Quesnel-Vallieres, Yoseph Barash, Alessandro Gardini
bioRxiv 2020.01.24.916015; doi: https://doi.org/10.1101/2020.01.24.916015
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Rapid and scalable profiling of nascent RNA with fastGRO
Elisa Barbieri, Connor Hill, Mathieu Quesnel-Vallieres, Yoseph Barash, Alessandro Gardini
bioRxiv 2020.01.24.916015; doi: https://doi.org/10.1101/2020.01.24.916015

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