Journal Article

Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq)

Core, Leighton J, Mahat, Dig Bijay, Waters, Colin T, Jonkers, Iris H, Lis, John T, Booth, Gregory T, Patel, Ravi K, Danko, Charles G, Kwak, Hojoong, Munson, Katie

2016

Overview

Mahat et al . describe how to map the genome-wide positions of active RNA polymerases using a modified nuclear run-on approach called PRO-seq. Details for PRO-cap, a modification that identifies transcription start sites, are also included. 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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 38/23 / 38/39 / 38/91 / 631/1647/2017 / 631/1647/514/1949 / 631/208/212/2019 / 631/337/572/2102 / Analysis / Analytical Chemistry / Animals / Base Pairing / Biological Techniques / Biotin / Chromosome Mapping - methods / Computational Biology/Bioinformatics / DNA-Directed RNA Polymerases - metabolism / Drosophila melanogaster - enzymology / Drosophila melanogaster - genetics / Genetic transcription / Genome-wide association studies / Genomes / Humans / Life Sciences / Mice / Microarrays / Organic Chemistry / Protocol / RNA - chemistry / RNA - genetics / RNA - metabolism / RNA polymerase / RNA polymerases / Sequence Analysis, RNA / Transcription Initiation Site / Yeasts