Bru-Seq
Bromouridine Sequencing
Bru-Seq maps nascent RNA transcripts using bromouridine tagging (Paulsen et al., 2013). Active RNAPII synthesizes RNA in the presence of Br-UTP. Tagged RNA transcripts are immunoseparated from total RNA using magnetic beads coated with anti-BrdU antibodies. The captured RNA transcripts are eluted and fragmented before synthesis of cDNA strands via RT and PCR amplification. The resultant cDNA strands are prepared for sequencing using an Illumina TruSeq RNA Library Prep Kit.
Advantages:
- Maps sequences of nascent RNA transcripts and determines relative transcription rate
- Detects lncRNAs (Paulsen et al., 2014)
- Detects transcription anywhere on the genome
Disadvantages:
- Limited to cell cultures and other artificial systems, due to the requirement for incubation in the presence of labeled nucleotides
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Andrade-Lima L., Veloso A. and Ljungman M. Transcription Blockage Leads to New Beginnings. Biomolecules. 2015;5:1600
References:
Lefkofsky H. B., Veloso A. and Ljungman M. Transcriptional and post-transcriptional regulation of nucleotide excision repair genes in human cells. Mutat Res. 2015;776:9-15
Kocab A. J., Veloso A., Paulsen M. T., Ljungman M. and Duckett C. S. Effects of physiological and synthetic IAP antagonism on c-IAP-dependent signaling. Oncogene. 2015;