Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

A rapid, sensitive, scalable method for Precision Run-On sequencing (PRO-seq)

View ORCID ProfileJulius Judd, Luke A. Wojenski, Lauren M. Wainman, View ORCID ProfileNathaniel D. Tippens, Edward J. Rice, Alexis Dziubek, Geno J. Villafano, View ORCID ProfileErin M. Wissink, Philip Versluis, Lina Bagepalli, Sagar R. Shah, Dig B. Mahat, Jacob M. Tome, Charles G. Danko, View ORCID ProfileJohn T. Lis, Leighton J. Core
doi: https://doi.org/10.1101/2020.05.18.102277
Julius Judd
1Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Julius Judd
Luke A. Wojenski
2Department of Molecular and Cell Biology, Institute of Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Lauren M. Wainman
2Department of Molecular and Cell Biology, Institute of Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nathaniel D. Tippens
1Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Nathaniel D. Tippens
Edward J. Rice
3Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Alexis Dziubek
1Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
2Department of Molecular and Cell Biology, Institute of Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Geno J. Villafano
2Department of Molecular and Cell Biology, Institute of Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Erin M. Wissink
1Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Erin M. Wissink
Philip Versluis
1Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Lina Bagepalli
1Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sagar R. Shah
1Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Dig B. Mahat
1Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jacob M. Tome
1Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Charles G. Danko
3Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
4Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
John T. Lis
1Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for John T. Lis
  • For correspondence: leighton.core@uconn.edu johnlis@cornell.edu
Leighton J. Core
2Department of Molecular and Cell Biology, Institute of Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: leighton.core@uconn.edu johnlis@cornell.edu
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Data/Code
  • Preview PDF
Loading

Abstract

Tracking active transcription with the nuclear run-on (NRO) assays has been instrumental in uncovering mechanisms of gene regulation. The coupling of NROs with high-throughput sequencing has facilitated the discovery of previously unannotated or undetectable RNA classes genome-wide. Precision run-on sequencing (PRO-seq) is a run-on variant that maps polymerase active sites with nucleotide or near-nucleotide resolution. One main drawback to this and many other nascent RNA detection methods is the somewhat intimidating multi-day workflow associated with creating the libraries suitable for high-throughput sequencing. Here, we present an improved PRO-seq protocol where many of the enzymatic steps are carried out while the biotinylated NRO RNA remains bound to streptavidin-coated magnetic beads. These adaptations reduce time, sample loss and RNA degradation, and we demonstrate that the resulting libraries are of the same quality as libraries generated using the original published protocol. The assay is also more sensitive which permits reproducible, high-quality libraries from 104–105 cells instead of 106–107. Altogether, the improved protocol is more tractable allows for nascent RNA profiling from small samples, such as rare samples or FACS sorted cell populations.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • https://github.com/JAJ256/qPRO

  • https://dx.doi.org/10.17504/protocols.io.57dg9i6

Copyright 
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 4.0 International license.
Back to top
PreviousNext
Posted May 19, 2020.
Download PDF
Data/Code
Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
A rapid, sensitive, scalable method for Precision Run-On sequencing (PRO-seq)
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
A rapid, sensitive, scalable method for Precision Run-On sequencing (PRO-seq)
Julius Judd, Luke A. Wojenski, Lauren M. Wainman, Nathaniel D. Tippens, Edward J. Rice, Alexis Dziubek, Geno J. Villafano, Erin M. Wissink, Philip Versluis, Lina Bagepalli, Sagar R. Shah, Dig B. Mahat, Jacob M. Tome, Charles G. Danko, John T. Lis, Leighton J. Core
bioRxiv 2020.05.18.102277; doi: https://doi.org/10.1101/2020.05.18.102277
Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
A rapid, sensitive, scalable method for Precision Run-On sequencing (PRO-seq)
Julius Judd, Luke A. Wojenski, Lauren M. Wainman, Nathaniel D. Tippens, Edward J. Rice, Alexis Dziubek, Geno J. Villafano, Erin M. Wissink, Philip Versluis, Lina Bagepalli, Sagar R. Shah, Dig B. Mahat, Jacob M. Tome, Charles G. Danko, John T. Lis, Leighton J. Core
bioRxiv 2020.05.18.102277; doi: https://doi.org/10.1101/2020.05.18.102277

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Genomics
Subject Areas
All Articles
  • Animal Behavior and Cognition (2646)
  • Biochemistry (5266)
  • Bioengineering (3678)
  • Bioinformatics (15796)
  • Biophysics (7253)
  • Cancer Biology (5627)
  • Cell Biology (8095)
  • Clinical Trials (138)
  • Developmental Biology (4765)
  • Ecology (7516)
  • Epidemiology (2059)
  • Evolutionary Biology (10576)
  • Genetics (7730)
  • Genomics (10131)
  • Immunology (5193)
  • Microbiology (13905)
  • Molecular Biology (5385)
  • Neuroscience (30779)
  • Paleontology (215)
  • Pathology (879)
  • Pharmacology and Toxicology (1524)
  • Physiology (2254)
  • Plant Biology (5022)
  • Scientific Communication and Education (1041)
  • Synthetic Biology (1385)
  • Systems Biology (4146)
  • Zoology (812)