4sUDRB-Seq

4sUDRB-Seq

4sUDRB-Seq is a sequencing method to investigate initiation frequencies and RNA elongation rates throughout the genome using 4-thiouridine (4sU) and 5,6-dichlor-obenzimidazole 1-β-d-ribofuranoside (DRB). Cells are first treated with DRB to inhibit RNA elongation and arrest RNA polymerase II (RNA pol II) at transcription start sites. The cells are then lysed, cleansed of DRB, and immediately incubated with 4sU to label newly transcribed RNA molecules. Biotin is added to bind with 4sU and are subsequently captured with streptavidin beads. Biotinylated RNA fragments are eluted and prepared for sequencing following the protocol in 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. Fuchs G., Rosenthal E., Bublik D.-R., Kaplan T. and Oren M. (2015) Gene body H2B monoubiquitylation regulates gene-selective RNA Polymerase II pause release and is not rate limiting for transcription elongation. bioRxiv