Global Run-On Sequencing (GRO-seq) Library Preparation from Drosophila Ovaries

  • Nikolay V. RozhkovEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1328)


In the past decade, deep-sequencing approaches have greatly improved our knowledge of the genome’s potential and have become a crucial milestone for new discoveries in genomics. Transcription is the first step of gene expression; therefore, the detection and measurement of transcription rates is of great interest. Here, a detailed protocol for global run-on sequencing (GRO-seq) library preparation from Drosophila ovaries is described. The method relies on rapid isolation of nuclei with halted transcription, then restarting transcription in physiological conditions in the presence of a labeled nucleotide. The newly transcribed nascent RNA is then isolated and cloned using a small RNA cloning protocol. Although it is time-consuming, the global run-on method allows the user to profile the position, orientation and amount of transcriptionally engaged RNA polymerases across the genome, therefore providing a snapshot of genome-wide transcription.

Key words

Drosophila Germline GRO-seq Nuclear Run-On Nascent RNA Transcriptome 



I thank J. Lis and A. Kalmykova laboratories for their efforts on improving GRO-seq and run-on protocols, respectively, which influenced the present work. I am grateful to Leah Sabin for critical reading of the manuscript, language editing, and helpful suggestions.


  1. 1.
    Zentner GE, Henikoff S (2014) High-resolution digital profiling of the epigenome. Nat Rev Genet 15:814–827CrossRefPubMedGoogle Scholar
  2. 2.
    Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Cech TR, Steitz JA (2014) The noncoding RNA revolution-trashing old rules to forge new ones. Cell 157:77–94CrossRefPubMedGoogle Scholar
  4. 4.
    Rinn JL, Chang HY (2012) Genome regulation by long noncoding RNAs. Annu Rev Biochem 81:145–166CrossRefPubMedGoogle Scholar
  5. 5.
    Sabin LR, Delas MJ, Hannon GJ (2013) Dogma derailed: the many influences of RNA on the genome. Mol Cell 49:783–794CrossRefPubMedGoogle Scholar
  6. 6.
    Core LJ, Waterfall JJ, Lis JT (2008) Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322:1845–1848PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Ingolia NT, Ghaemmaghami S, Newman JR et al (2009) Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324:218–223PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Batut P, Dobin A, Plessy C et al (2013) High-fidelity promoter profiling reveals widespread alternative promoter usage and transposon-driven developmental gene expression. Genome Res 23:169–180PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Chang H, Lim J, Ha M et al (2014) TAIL-seq: genome-wide determination of poly(A) tail length and 3′ end modifications. Mol Cell 53:1044–1052CrossRefPubMedGoogle Scholar
  10. 10.
    Kwak H, Fuda NJ, Core LJ et al (2013) Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339:950–953PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Churchman LS, Weissman JS (2012) Native elongating transcript sequencing (NET-seq). Curr Protoc Mol Biol Chapter 4;Unit 4, 14, 11–17Google Scholar
  12. 12.
    Khodor YL, Rodriguez J, Abruzzi KC et al (2011) Nascent-seq indicates widespread cotranscriptional pre-mRNA splicing in Drosophila. Genes Dev 25:2502–2512PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Paulsen MT, Veloso A, Prasad J et al (2014) Use of Bru-Seq and BruChase-Seq for genome-wide assessment of the synthesis and stability of RNA. Methods 67:45–54PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Rozhkov NV, Hammell M, Hannon GJ (2013) Multiple roles for Piwi in silencing Drosophila transposons. Genes Dev 27:400–412PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Core LJ, Waterfall JJ, Gilchrist DA et al (2012) Defining the status of RNA polymerase at promoters. Cell Rep 2:1025–1035PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Chopra VS, Hendrix DA, Core LJ et al (2011) The polycomb group mutant esc leads to augmented levels of paused Pol II in the Drosophila embryo. Mol Cell 42:837–844PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Larschan E, Bishop EP, Kharchenko PV et al (2011) X chromosome dosage compensation via enhanced transcriptional elongation in Drosophila. Nature 471:115–118PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Mohn F, Sienski G, Handler D et al (2014) The rhino-deadlock-cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila. Cell 157:1364–1379CrossRefPubMedGoogle Scholar
  19. 19.
    Hafner M, Renwick N, Farazi TA et al (2012) Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing. Methods 58:164–170PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    McGinn J, Czech B (2014) Small RNA library construction for high-throughput sequencing. Methods Mol Biol 1093:195–208CrossRefPubMedGoogle Scholar
  21. 21.
    Shpiz S, Olovnikov I, Sergeeva A et al (2011) Mechanism of the piRNA-mediated silencing of Drosophila telomeric retrotransposons. Nucleic Acids Res 39:8703–8711PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Sigova A, Vagin V, Zamore PD (2006) Measuring the rates of transcriptional elongation in the female Drosophila melanogaster germ line by nuclear run-on. Cold Spring Harb Symp Quant Biol 71:335–341CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Cold Spring Harbor LaboratoryCold Spring HarborUSA

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