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Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq)
Open accessAbstract
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.
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Metadata
- Authors
- Mahat, D. B., Kwak, H., Booth, G. T., Jonkers, I. H., Danko, C. G., Patel, R. K., Waters, C. T., Munson, K., Core, L. J., & Lis, J. T.
- Venue
- Nature Protocols
- Published
- 2016-07-21
- Volume
- 11
- Issue
- 8
- Pages
- 1455-1476
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W2498392978
- Cited by
- 508
- References
- 37
- FWCI
- 12.64
- Type
- journal article
- Language
- en
- Open access
- green
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