BruDRB-Seq

BruDRB-Seq

BruDRB-Seq reports the elongation rate of RNAPII. 5,6-dichlorobenzimidazole 1-beta-D-ribofuranoside (DRB) is added to cells before elongation to inhibit RNAPII transiently, allowing synchronized transcriptional initiation throughout the genome. Upon removal of DRB, Br-UTP is added instead of UTP, along with other nucleotides. After cell lysis, RNA is isolated and fragmented. Next, bromouridine-tagged RNA is immunoseparated from total RNA using magnetic beads coated with anti-BrdU antibodies. cDNA libraries are generated, using the TruSeq RNA library preparation protocol, and then sequenced..

Pros:

• Quantifies RNA elongation rate throughout the whole genome
• Newly transcribed RNA molecules are tagged through their entire length

Cons:

• Limited to cell cultures and other artificial systems, due to the requirement for incubation in the presence of labeled nucleotides
• Bru-Seq must be performed in conjunction with Bru-DRB-Seq for accurate data analysis

• BruDRB-Seq: Veloso A., Kirkconnell K. S., Magnuson B., Biewen B., Paulsen M. T., et al. (2014) Rate of elongation by RNA polymerase II is associated with specific gene features and epigenetic modifications. Genome Res 24: 896-905

1. 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. Fuchs G., Voichek Y., Rabani M., Benjamin S., Gilad S., et al. (2015) Simultaneous measurement of genome-wide transcription elongation speeds and rates of RNA polymerase II transition into active elongation with 4sUDRB-seq. Nat Protoc 10: 605-618