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

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

Global run-on sequencing (GRO-Seq ) and 5’-Bromo-uridine immunoprecipitation chase (BRIC-seq , Bru-Seq , BruChase-Seq) map binding sites of transcriptionally active RNA polymerase II. In these methods, active RNA polymerase II is allowed to run on in the presence of Br-UTP. RNAs are hydrolyzed and purified using beads coated with Brd-UTP antibody. The eluted RNA undergoes cap removal and end repair prior to reverse transcription to cDNA. Deep sequencing of the cDNA provides sequences of RNAs that are actively transcribed by RNA polymerase II.

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

Cons:

• The protocol is 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
• New initiation events may occur during the run-on step
• Physical impediments may block the polymerases

• 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. Andrade-Lima L. C., Veloso A., Paulsen M. T., Menck C. F. and Ljungman M. (2015) DNA repair and recovery of RNA synthesis following exposure to ultraviolet light are delayed in long genes. Nucleic Acids Res 43: 2744-2756 
2. Danko C. G., Hyland S. L., Core L. J., Martins A. L., Waters C. T., et al. (2015) Identification of active transcriptional regulatory elements from GRO-seq data. Nat Methods 12: 433-438
3. Leveille N., Melo C. A., Rooijers K., Diaz-Lagares A., Melo S. A., et al. (2015) Genome-wide profiling of p53-regulated enhancer RNAs uncovers a subset of enhancers controlled by a lncRNA. Nat Commun 6: 6520
4. Madsen J. G., Schmidt S. F., Larsen B. D., Loft A., Nielsen R., et al. (2015) iRNA-seq: computational method for genome-wide assessment of acute transcriptional regulation from total RNA-seq data. Nucleic Acids Res 43: e40
5. Williams L. H., Fromm G., Gokey N. G., Henriques T., Muse G. W., et al. (2015) Pausing of RNA Polymerase II Regulates Mammalian Developmental Potential through Control of Signaling Networks. Mol Cell 58: 311-322