4sUDRB-Seq

4sUDRB-Seq

4sUDRB-Seq investigates initiation frequencies and RNA elongation rates throughout the genome using 4-thiouridine (4-SU) and DRB. Cells are first treated with DRB to inhibit RNA elongation and arrest RNAPII at TSS. The cells are lysed, cleansed of DRB, and immediately incubated with 4-SU to label newly transcribed RNA molecules. Biotin is added to bind with 4-SU, and the tagged RNA is subsequently captured using streptavidin beads. The biotinylated RNA fragments are eluted and prepared for sequencing according to the protocol in the TruSeq RNA Library Preparation Kit.

Pros:

• Measures RNA elongation rate and transcription initiation rate simultaneously
• Sequencing reads behave dynamically throughout the transcription wave – useful in accurately determining transcription initiation rate¹
• No previous knowledge of the sequence is needed

Cons:

• High toxicity of 4sU can debilitate experiments with slow elongation rates¹
• Measuring genome-wide transition of RNA pol II into active elongation can be cost-inefficient due to the high sequencing depth needed¹
• Dynamic sequence reads makes identification of transcripts ends challenging¹
• Limited to cultured cells

• 4sUDRB-Seq: Fuchs G., Voichek Y., Benjamin S., Gilad S., Amit I., et al. (2014) 4sUDRB-seq: measuring genomewide transcriptional elongation rates and initiation frequencies within cells. Genome Biol 15: R69
• 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

1. Rabani M., Raychowdhury R., Jovanovic M., et al. High-Resolution Sequencing and Modeling Identifies Distinct Dynamic RNA Regulatory Strategies. Cell. 159:1698-1710
2. Duffy E. E., Rutenberg-Schoenberg M., Stark C. D., Kitchen R. R., Gerstein M. B. and Simon M. D. Tracking Distinct RNA Populations Using Efficient and Reversible Covalent Chemistry. Mol Cell. 2015;59:858-866
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. Zhang Y., Xue W., Li X., et al. The Biogenesis of Nascent Circular RNAs. Cell Rep. 2016;15:611-624
2. Jaenicke L. A., von Eyss B., Carstensen A., et al. Ubiquitin-Dependent Turnover of MYC Antagonizes MYC/PAF1C Complex Accumulation to Drive Transcriptional Elongation. Mol Cell. 2016;61:54-67
3. Fuchs G., Rosenthal E., Bublik D.-R., Kaplan T. and Oren M. Gene body H2B monoubiquitylation regulates gene-selective RNA Polymerase II pause release and is not rate limiting for transcription elongation. bioRxiv. 2015;