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

Nature Protocols volume 10, pages 605–618 (2015)Cite this article

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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.

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Figure 1: Schematic diagram of 4sUDRB-seq.
Figure 2: Schematic comparison of 4sUDRB-seq and GRO-seq-based methods.
Figure 3: Representative results from DRB treatment and removal.
Figure 4: Representative results from 4sU-tagged enrichment.
Figure 5: Representative results from 4sUDRB-seq.

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Acknowledgements

We thank D.R. Bublik, E. Kotler and L. Golomb for helpful discussions. This work was supported in part by grant 293438 (RUBICAN) from the European Research Council and the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation. M.O. is an incumbent of the Andre Lwoff chair in molecular biology.

Author information

Authors and Affiliations

  1. Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

    Gilad Fuchs & Moshe Oren

  2. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

    Yoav Voichek

  3. Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology and The Broad Institute, Cambridge

    Michal Rabani

  4. Massachusetts, USA

    Michal Rabani

  5. The Israel National Center for Personalized Medicine (INCPM), Weizmann Institute of Science, Rehovot, Israel

    Sima Benjamin & Shlomit Gilad

  6. Department of Immunology, Weizmann Institute of Science, Rehovot, Israel

    Ido Amit

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  1. Gilad Fuchs
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Contributions

G.F., M.R. and M.O. developed the protocol; G.F., Y.V. and M.O. wrote the manuscript; Y.V., S.B., S.G. and I.A. contributed to protocol development.

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Correspondence to Moshe Oren.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1: Primer sequences.

List of primers and their sequences used for qRT-PCR validation (Steps 16 and 62) examples shown in Figures 3 and 4. (XLSX 11 kb)

Supplementary Software: Scripts for bioinformatics analysis.

Compressed file containing the scripts: reads2chr.cc and find_boundary_4sUDRBseq.m, needed for the bioinformatics analysis (Steps 66-72). (ZIP 3 kb)

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Fuchs, G., Voichek, Y., Rabani, M. et al. 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 (2015). https://doi.org/10.1038/nprot.2015.035

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