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.

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Figure 1: Flowchart of the PRO-seq and PRO-cap protocol.
Figure 2: Gel images of library products.
Figure 3: Genome browser examples of PRO-seq and PRO-cap results.

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Acknowledgements

We thank all the present and former members of the Lis lab who provided advice and support for this work. We especially thank N. Fuda, a former member of our lab, for help at many steps of this protocol. Research reported in this publication was supported by National Institutes of Health (NIH) grant R01GM25232 to J.T.L. and European Research Council Advanced Grant ERCadv-671274 to I.H.J. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

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H.K., L.J.C. and J.T.L. conceived the method and designed the experiments. H.K. and D.B.M. carried out the experiments that generated the data. G.T.B., I.H.J., R.K.P., C.T.W., K.M. and L.J.C. carried out experiments that optimized the protocol. C.G.D. contributed in generating the pipelines for the computational analysis. H.K., D.B.M. and J.T.L. wrote the manuscript.

Corresponding authors

Correspondence to Leighton J Core or John T Lis.

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The authors declare no competing financial interests.

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Mahat, D., Kwak, H., Booth, G. et al. Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). Nat Protoc 11, 1455–1476 (2016). https://doi.org/10.1038/nprot.2016.086

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