GRO-Seq/BRIC-Seq/Bru-Seq/BruChase-Seq

GRO-Seq/BRIC-Seq/Bru-Seq/BruChase-Seq

GRO-Seq maps the binding sites of transcriptionally active RNA polymerase II (RNAPII). 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.

Pros:

• 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 RNAs¹

Cons:

• 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 nucle²i .
• New initiation events may occur during the run-on step
• Physical impediments may block the polymerases
• Resolution is only 30–100 nt³
• Requires nascent RNAs of at least 18 nt⁴

• GRO-Seq: Core L. J., Waterfall J. J. and Lis J. T. (2008) Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322: 1845-1848
• BRIC-Seq: Tani H., Mizutani R., Salam K. A., Tano K., Ijiri K., et al. (2012) Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals. Genome Res 22: 947-956 
• Bru-Seq / BruChase-Seq: Paulsen M. T., Veloso A., Prasad J., Bedi K., Ljungman E. A., et al. (2013) Coordinated regulation of synthesis and stability of RNA during the acute TNF-induced proinflammatory response. Proc Natl Acad Sci U S A 110: 2240-2245
• 1. 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
• 2. Adelman K. and Lis J. T. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans. Nat Rev Genet. 2012;13:720-731
• 3. 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
• 4. Mayer A. and Churchman L. S. Genome-wide profiling of RNA polymerase transcription at nucleotide resolution in human cells with native elongating transcript sequencing. Nat Protoc. 2016;11:813-833

1. 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
2. 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
3. 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

1. 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
2. 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
3. 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
4. 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
5. 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
6. 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
7. 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
8. 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
9. 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
10. Petryk N., Kahli M., d'Aubenton-Carafa Y., et al. Replication landscape of the human genome. Nat Commun. 2016;7:10208
11. 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