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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References
Fuda, N.J., Ardehali, M.B. & Lis, J.T. Defining mechanisms that regulate RNA polymerase II transcription in vivo. Nature 461, 186–192 (2009).
Adelman, K. & Lis, J.T. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans. Nat. Rev. Genet. 13, 720–731 (2012).
Core, L.J. et al. Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers. Nat. Genet. 46, 1311–1320 (2014).
Heinz, S., Romanoski, C.E., Benner, C. & Glass, C.K. The selection and function of cell type-specific enhancers. Nat. Rev. Mol. Cell Biol. 16, 144–154 (2015).
Vahedi, G. et al. Super-enhancers delineate disease-associated regulatory nodes in T cells. Nature 520, 558–562 (2015).
Churchman, L.S. & Weissman, J.S. Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 469, 368–373 (2011).
Larson, M.H. et al. A pause sequence enriched at translation start sites drives transcription dynamics in vivo. Science 344, 1042–1047 (2014).
Nojima, T. et al. Mammalian NET-Seq reveals genome-wide nascent transcription coupled to RNA processing. Cell 161, 526–540 (2015).
Weber, C.M., Ramachandran, S. & Henikoff, S. Nucleosomes are context-specific, H2A.Z-modulated barriers to RNA polymerase. Mol. Cell. 53, 819–830 (2014).
Core, L.J., Waterfall, J.J. & Lis, J.T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008).
Kwak, H., Fuda, N.J., Core, L.J. & Lis, J.T. Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339, 950–953 (2013).
Jonkers, I., Kwak, H. & Lis, J.T. Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons. Elife 3, e02407 (2014).
Kwak, H. & Lis, J.T. Control of transcriptional elongation. Annu. Rev. Genet. 47, 483–508 (2013).
Hah, N. et al. A rapid, extensive, and transient transcriptional response to estrogen signaling in breast cancer cells. Cell 145, 622–634 (2011).
Min, I.M. et al. Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells. Genes Dev. 25, 742–754 (2011).
Larschan, E. et al. X chromosome dosage compensation via enhanced transcriptional elongation in Drosophila. Nature 471, 115–118 (2011).
Cock, P.J.A., Fields, C.J., Goto, N., Heuer, M.L. & Rice, P.M. The Sanger FASTQ file format for sequences with quality scores, and the Solexa/Illumina FASTQ variants. Nucl. Acids Res. 38, 1767–1771 (2010).
Core, L.J. et al. Defining the status of RNA polymerase at promoters. Cell Rep. 2, 1025–1035 (2012).
Seila, A.C. et al. Divergent transcription from active promoters. Science 322, 1849–1851 (2008).
Carninci, P. et al. High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Genomics 37, 327–336 (1996).
Andersson, R. et al. An atlas of active enhancers across human cell types and tissues. Nature 507, 455–461 (2014).
Forrest, A.R.R. et al. A promoter-level mammalian expression atlas. Nature 507, 462–470 (2014).
Wang, D. et al. Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature 474, 390–394 (2011).
Hah, N., Murakami, S., Nagari, A., Danko, C.G. & Kraus, W.L. Enhancer transcripts mark active estrogen receptor binding sites. Genome Res. 23, 1210–1223 (2013).
Fejes-Toth, K. et al. Post-transcriptional processing generates a diversity of 5-modified long and short RNAs. Nature 457, 1028–1032 (2009).
Rhee, H.S. & Pugh, B.F. Genome-wide structure and organization of eukaryotic pre-initiation complexes. Nature 483, 295–301 (2012).
Li, J. et al. Kinetic competition between elongation rate and binding of NELF controls promoter-proximal pausing. Mol. Cell 50, 711–722 (2013).
Mayer, A. et al. Native elongating transcript sequencing reveals human transcriptional activity at nucleotide resolution. Cell 161, 541–554 (2015).
Mahat, D.B., Salamanca, H.H., Duarte, F.M., Danko, C.G. & Lis, J.T. Mammalian heat shock response and mechanisms underlying its genome-wide transcriptional regulation. Mol. Cell 62, 63–78 (2016).
García-Martínez, J., Aranda, A. & Pérez-Ortín, J.E. Genomic run-on evaluates transcription rates for all yeast genes and identifies gene regulatory mechanisms. Mol. Cell 15, 303–313 (2004).
Collart, M.A. & Oliviero, S. Preparation of yeast RNA. Curr. Protoc. Mol. Biol. Chapter 13, Unit13.12 (2001).
Job, D. et al. Complex RNA chain elongation kinetics by wheat germ RNA polymerase II. Nucleic Acids Res. 12, 3303–3319 (1984).
Fu, G.K. et al. Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations. Proc. Natl. Acad. Sci. USA 111, 1891–1896 (2014).
Marcel, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Quinlan, A.R. & Hall, I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
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.
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Mahat, D., Kwak, H., Booth, G. et al. Base-pair-resolution genome-wide map** 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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DOI: https://doi.org/10.1038/nprot.2016.086
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