Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide

Nature Protocols volume 11, pages 413–428 (2016)Cite this article

Subjects

Abstract

The transcription cycle of RNA polymerase II (Pol II) correlates with changes to the phosphorylation state of its large subunit C-terminal domain (CTD). We recently developed Native Elongation Transcript sequencing using mammalian cells (mNET-seq), which generates single-nucleotide–resolution genome-wide profiles of nascent RNA and co-transcriptional RNA processing that are associated with different CTD phosphorylation states. Here we provide a detailed protocol for mNET-seq. First, Pol II elongation complexes are isolated with specific phospho-CTD antibodies from chromatin solubilized by micrococcal nuclease digestion. Next, RNA derived from within the Pol II complex is size fractionated and Illumina sequenced. Using mNET-seq, we have previously shown that Pol II pauses at both ends of protein-coding genes but with different CTD phosphorylation patterns, and we have also detected phosphorylation at serine 5 (Ser5-P) CTD-specific splicing intermediates and Pol II accumulation over co-transcriptionally spliced exons. With moderate biochemical and bioinformatic skills, mNET-seq can be completed in ∼6 d, not including sequencing and data analysis.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Overview of the mNET-seq procedure.
Figure 2: Examples of gel purification steps in the mNET-seq method.
Figure 3: Example of a mNET-seq profile for the SIK1 gene.

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Ozsolak, F. & Milos, P.M. RNA sequencing: advances, challenges and opportunities. Nat. Rev. Genet. 12, 87–98 (2011).

    Article  CAS  Google Scholar 

  2. Wilhelm, B.T. & Landry, J.R. RNA-seq-quantitative measurement of expression through massively parallel RNA-sequencing. Methods 48, 249–257 (2009).

    Article  CAS  Google Scholar 

  3. Moore, M.J. & Proudfoot, N.J. Pre-mRNA processing reaches back to transcription and ahead to translation. Cell 136, 688–700 (2009).

    Article  CAS  Google Scholar 

  4. Morlando, M. et al. Primary microRNA transcripts are processed co-transcriptionally. Nat. Struct. Mol. Biol. 15, 902–909 (2008).

    Article  CAS  Google Scholar 

  5. Pawlicki, J.M. & Steitz, J.A. Primary microRNA transcript retention at sites of transcription leads to enhanced microRNA production. J. Cell Biol. 182, 61–76 (2008).

    Article  CAS  Google Scholar 

  6. Heidemann, M., Hintermair, C., Voss, K. & Eick, D. Dynamic phosphorylation patterns of RNA polymerase II CTD during transcription. Biochim. Biophys. Acta. 1829, 55–62 (2013).

    Article  CAS  Google Scholar 

  7. Core, L.J. & Lis, J.T. Transcription regulation through promoter-proximal pausing of RNA polymerase II. Science 319, 1791–1792 (2008).

    Article  CAS  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

  9. Weber, C.M., Ramachandran, S. & Henikoff, S. Nucleosomes are context-specific, H2A.Z-modulated barriers to RNA polymerase. Mol. Cell 53, 819–830 (2014).

    Article  CAS  Google Scholar 

  10. Mayer, A. et al. Native elongating transcript sequencing reveals human transcriptional activity at nucleotide resolution. Cell 161, 541–554 (2015).

    Article  CAS  Google Scholar 

  11. Churchman, L.S. & Weissman, J.S. Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 469, 368–373 (2011).

    Article  CAS  Google Scholar 

  12. Larson, M.H. et al. A pause sequence enriched at translation start sites drives transcription dynamics in vivo. Science 344, 1042–1047 (2014).

    Article  CAS  Google Scholar 

  13. Nojima, T. et al. Mammalian NET-seq reveals genome-wide nascent transcription coupled to RNA processing. Cell 161, 526–540 (2015).

    Article  CAS  Google Scholar 

  14. Wuarin, J. & Schibler, U. Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing. Mol. Cell Biol. 14, 7219–7225 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Dye, M.J., Gromak, N. & Proudfoot, N.J. Exon tethering in transcription by RNA polymerase II. Mol. Cell 21, 849–859 (2006).

    Article  CAS  Google Scholar 

  16. West, S., Proudfoot, N.J. & Dye, M.J. Molecular dissection of mammalian RNA polymerase II transcriptional termination. Mol. Cell 29, 600–610 (2008).

    Article  CAS  Google Scholar 

  17. Nojima, T., Dienstbier, M., Murphy, S., Proudfoot, N.J. & Dye, M.J. Definition of RNA polymerase II CoTC terminator elements in the human genome. Cell Rep. 3, 1080–1092 (2013).

    Article  CAS  Google Scholar 

  18. Storvall, H., Ramskold, D. & Sandberg, R. Efficient and comprehensive representation of uniqueness for next-generation sequencing by minimum unique length analyses. PLoS ONE 8, e53822 (2013).

    Article  CAS  Google Scholar 

  19. Martinez-Rucobo, F.W. et al. Molecular basis of transcription-coupled pre-mRNA capping. Mol. Cell 58, 1079–1089 (2015).

    Article  CAS  Google Scholar 

  20. 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).

    Article  CAS  Google Scholar 

  21. Min, I.M. et al. Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells. Genes Dev. 25, 742–754 (2011).

    Article  CAS  Google Scholar 

  22. Saunders, A., Core, L.J., Sutcliffe, C., Lis, J.T. & Ashe, H.L. Extensive polymerase pausing during Drosophila axis patterning enables high-level and pliable transcription. Genes Dev. 27, 1146–1158 (2013).

    Article  CAS  Google Scholar 

  23. Kruesi, W.S., Core, L.J., Waters, C.T., Lis, J.T. & Meyer, B.J. Condensin controls recruitment of RNA polymerase II to achieve nematode X-chromosome dosage compensation. Elife 2, e00808 (2013).

    Article  Google Scholar 

  24. 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).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Murphy for critically testing this protocol, and A.R. Fialho Grosso for advice on bioinformatics analysis. This work was supported by funding to N.J.P. (Wellcome Trust Programme no. 091805/Z/10/Z and European Research Council (ERC) Advanced grant no. 339270) and to M.C-F. (Fundação Ciência e Tecnologia, Portugal).

Author information

Authors and Affiliations

  1. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

    Takayuki Nojima & Nicholas J Proudfoot

  2. Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal

    Tomás Gomes & Maria Carmo-Fonseca

Authors
  1. Takayuki Nojima
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tomás Gomes
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maria Carmo-Fonseca
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nicholas J Proudfoot
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.N., M.C.-F. and N.J.P. designed the protocol. All authors wrote the paper. T.N. developed the protocol and performed all experiments. T.G. bioinfomatically analyzed the data.

Corresponding authors

Correspondence to Takayuki Nojima or Nicholas J Proudfoot.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 mNET-seq vs GRO-seq

Correlation between log2 (RPM) of Total Pol II mNET-seq and GRO-seq data for genome windows of 500kb. Both samples show very good correlation (Pearson's Correlation Coeficient (PCC) = 0.93, p-value < 2.2e-16). GRO-seq data (GEO accession number GSM1518913) and RNA-seq data used to determine gene expression (GEO accession number GSM1155630) were from previously published studies (Andersson, R. et al. Nat. Commun. 5, 5336 (2014); Lacoste, N. et al. Mol. Cell 53, 631-644 (2014)).

Supplementary Figure 2 mNET-seq Pol II specificity

Boxplot distribution comparison of log2 reads per base (Rpb) values for Total RNA Pol II mNET-seq in expressed protein coding, tRNA and rRNA genes. For protein coding genes, only the region [TSS, TSS+100] is used, to consider a region without reads corresponding to splicing intermediates (which appear at the end of exons), and also to use a region with a size similar to that of tRNA and rRNA genes to avoid any size bias when normalizing.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2 (PDF 237 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Nojima, T., Gomes, T., Carmo-Fonseca, M. et al. Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide. Nat Protoc 11, 413–428 (2016). https://doi.org/10.1038/nprot.2016.012

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2016.012

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing