GRO-seq

Global Run-on Sequencing

GRO-Seq maps the binding sites of transcriptionally active RNA polymerase II (RNAPII) (Core et al., 2008). In this method, active RNAPII is allowed to run on in the presence of 5-bromouridine 5′-triphosphate (Br-UTP). RNAs are hydrolyzed and purified using beads coated with antibodies to 5-bromo-2-deoxyuridine (BrdU). After cap removal and end repair, the eluted RNA is reverse-transcribed to cDNA. Deep sequencing of the cDNA identifies RNAs that are actively transcribed by RNAPII.

Advantages:

• Maps position of transcriptionally engaged RNA polymerases
• Determines relative activity of transcription sites
• Detects sense and antisense transcription
• Detects transcription anywhere on the genome
• No prior knowledge of transcription sites is needed
• Provides robust coverage of enhancer- and promoter-associated RNAs (Melnik et al., 2016)

Disadvantages:

• Limited to cell cultures and other artificial systems, due to the requirement for incubation in the presence of labeled nucleotides
• Artifacts may be introduced during the preparation of the nuclei (Adelman et al., 2012)
• New initiation events may occur during the run-on step
• Physical impediments may block the polymerases
• Resolution is only 30_100 nt (Nojima et al., 2016)
• Requires nascent RNAs of at least 18 nt (Mayer et al., 2016).

Reagents:

Illumina Library prep and Array Kit Selector



Reviews:

Murakawa Y., Yoshihara M., Kawaji H., Nishikawa M., Zayed H., et al. Enhanced Identification of Transcriptional Enhancers Provides Mechanistic Insights into Diseases. Trends Genet. 2016;32:76-88

Li Y., Chen C. Y., Kaye A. M. and Wasserman W. W. The identification of cis-regulatory elements: A review from a machine learning perspective. Biosystems. 2015;138:6-17

Jonkers I. and Lis J. T. Getting up to speed with transcription elongation by RNA polymerase II. Nat Rev Mol Cell Biol. 2015;16:167-177



References:

Schwer B., Wei P. C., Chang A. N., et al. Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor cells. Proc Natl Acad Sci U S A. 2016;113:2258-2263

Chen Y. C., Stuwe E., Luo Y., et al. Cutoff Suppresses RNA Polymerase II Termination to Ensure Expression of piRNA Precursors. Mol Cell. 2016;63:97-109

Czimmerer Z., Varga T., Kiss M., et al. The IL-4/STAT6 signaling axis establishes a conserved microRNA signature in human and mouse macrophages regulating cell survival via miR-342-3p. Genome Med. 2016;8:63

Day D. S., Zhang B., Stevens S. M., et al. Comprehensive analysis of promoter-proximal RNA polymerase II pausing across mammalian cell types. Genome Biol. 2016;17:120

de Dieuleveult M., Yen K., Hmitou I., et al. Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells. Nature. 2016;530:113-116

Flynn R. A., Do B. T., Rubin A. J., Calo E., Lee B., et al. 7SK-BAF axis controls pervasive transcription at enhancers. Nat Struct Mol Biol. 2016;23:231-238

Korkmaz G., Lopes R., Ugalde A. P., et al. Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9. Nat Biotechnol. 2016;34:192-198

Melnik S., Caudron-Herger M., Brant L., et al. Isolation of the protein and RNA content of active sites of transcription from mammalian cells. Nat Protoc. 2016;11:553-565

Nojima T., Gomes T., Carmo-Fonseca M. and Proudfoot N. J. Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide. Nat Protoc. 2016;11:413-428

Petryk N., Kahli M., d’Aubenton-Carafa Y., et al. Replication landscape of the human genome. Nat Commun. 2016;7:10208

Woolnough J. L., Atwood B. L., Liu Z., Zhao R. and Giles K. E. The Regulation of rRNA Gene Transcription during Directed Differentiation of Human Embryonic Stem Cells. PLoS One. 2016;11:e0157276

Edit: GRO-seq

You must be logged in to edit this entry.

History: GRO-seq

Revision by sbrumpton on 2017-06-21 07:50:23 - Show/Hide