title: Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq).
abstract: We provide a protocol for precision nuclear run-on sequencing (PRO-seq) and its variant, PRO-cap, which map the location of active RNA polymerases (PRO-seq) or transcription start sites (TSSs) (PRO-cap) genome-wide at high resolution. The density of RNA polymerases at a particular genomic locus directly reflects the level of nascent transcription at that region. Nuclei are isolated from cells and, under nuclear run-on conditions, transcriptionally engaged RNA polymerases incorporate one or, at most, a few biotin-labeled nucleotide triphosphates (biotin-NTPs) into the 3' end of nascent RNA. The biotin-labeled nascent RNA is used to prepare sequencing libraries, which are sequenced from the 3' end to provide high-resolution positional information for the RNA polymerases. PRO-seq provides much higher sensitivity than ChIP-seq, and it generates a much larger fraction of usable sequence reads than ChIP-seq or NET-seq (native elongating transcript sequencing). Similarly to NET-seq, PRO-seq maps the RNA polymerase at up to base-pair resolution with strand specificity, but unlike NET-seq it does not require immunoprecipitation. With the protocol provided here, PRO-seq (or PRO-cap) libraries for high-throughput sequencing can be generated in 4-5 working days. The method has been applied to human, mouse, Drosophila melanogaster and Caenorhabditis elegans cells and, with slight modifications, to yeast.
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: Dig Bijay
authors.initials: DB
authors.lastname: Mahat
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: Hojoong
authors.initials: H
authors.lastname: Kwak
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: Gregory T
authors.initials: GT
authors.lastname: Booth
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: Iris H
authors.initials: IH
authors.lastname: Jonkers
authors.affiliation: The Baker Institute of Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.
authors.forename: Charles G
authors.identifier: 0000-0002-1999-7125
authors.initials: CG
authors.lastname: Danko
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: Ravi K
authors.initials: RK
authors.lastname: Patel
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: Colin T
authors.initials: CT
authors.lastname: Waters
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: Katie
authors.identifier: 0000-0001-8413-6498
authors.initials: K
authors.lastname: Munson
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: Leighton J
authors.initials: LJ
authors.lastname: Core
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: John T
authors.initials: JT
authors.lastname: Lis
mesh_terms: D000818:Animals; D020029:Base Pairing; D002874:Chromosome Mapping / Q000379:methods*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics; D006801:Humans; D051379:Mice; D012313:RNA / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017423:Sequence Analysis, RNA; D024363:Transcription Initiation Site
chemical_list: D012313:RNA; D012321:DNA-Directed RNA Polymerases