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Nat Protoc. 2015 Apr;10(4):605-18. doi: 10.1038/nprot.2015.035. Epub 2015 Mar 26.

Simultaneous measurement of genome-wide transcription elongation speeds and rates of RNA polymerase II transition into active elongation with 4sUDRB-seq.

Author information

1
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.
2
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
3
Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology and The Broad Institute, Cambridge, Massachusetts, USA.
4
The Israel National Center for Personalized Medicine (INCPM), Weizmann Institute of Science, Rehovot, Israel.
5
Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

Abstract

4sUDRB-seq separately measures, on a genomic scale, the distinct contributions of transcription elongation speed and rate of RNA polymerase II (Pol II) transition into active elongation (TAE) to the overall mRNA production rate. It uses reversible inhibition of transcription elongation with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), combined with a pulse of 4-thiouridine (4sU), to tag newly transcribed RNA. After DRB removal, cells are collected at several time points, and tagged RNA is biotinylated, captured on streptavidin beads and sequenced. 4sUDRB-seq enables the comparison of elongation speeds between different developmental stages or different cell types, and it allows the impact of specific transcription factors on transcription elongation speed versus TAE to be studied. RNA preparation takes ∼4 d to complete, with deep sequencing requiring an additional ∼4-11 d plus 1-3 d for bioinformatics analysis. The experimental protocol requires basic molecular biology skills, whereas data analysis requires knowledge in bioinformatics, particularly MATLAB and the Linux environment.

PMID:
25811895
DOI:
10.1038/nprot.2015.035
[Indexed for MEDLINE]

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