Abstract
The dynamics of transcription can be studied genome wide by high-throughput sequencing of nascent and newly synthesized RNA. 4-thiouridine (4SU) labeling in vivo enables the specific capture of such new transcripts, with 4SU residues being tagged by biotin linkers and captured using streptavidin beads before library production and high-throughput sequencing. To achieve high-resolution profiles of transcribed regions, an RNA fragmentation step before biotin tagging was introduced, in an approach known as transient transcriptome sequencing (TT-seq). We recently introduced a chemical approach for RNA fragmentation that we refer to as TTchem-seq. We describe how TTchem-seq can be used in combination with transient inhibition of early elongation using the reversible CDK9 inhibitor, 5,6-dichlorobenzimidazole 1-β-d-ribofuranoside (DRB), to measure RNA polymerase II (RNAPII) elongation rates in vivo, a technique we call DRB/TTchem-seq. Here, we provide detailed protocols for carrying out TTchem-seq and DRB/TTchem-seq, including computational analysis. Experiments and data analysis can be performed over a period of 10–13 d and require molecular biology and bioinformatics skills.
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Data availability
All sequencing data are available under GEO no. GSE121826.
Code availability
All code used to analyze TTchem-seq and DRB/TTchem-seq is available at https://github.com/crickbabs/DRB_TT-seq/releases/tag/v1.2 and https://github.com/crickbabs/DRB_TT-seq.
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Acknowledgements
This work was supported by the Francis Crick Institute (which receives its core funding from Cancer Research UK (FC001166), the UK Medical Research Council (FC001166) and the Wellcome Trust (FC001166)) and by a grant from the European Research Council (agreement 693327 (TRANSDAM)). L.H.G. was supported by the EMBO-LTF program (EMBO ALTF 1026-2014). We thank A. Herlihy for helpful comments on the manuscript.
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L.H.G. and J.Q.S. conceived the experimental setup. L.H.G. performed all optimization steps and experiments. R.M. developed the bioinformatics pipeline for analysis of TTchem-seq and DRB/TTchem-seq. J.Q.S. supervised various aspects of the work. L.H.G. and J.Q.S wrote the manuscript with assistance from R.M.
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Peer review information Nature Protocols thanks Didier Devys and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key reference using this protocol
Gregersen, L. H. et al. Cell 177, 1797–1813.e18 (2019): https://doi.org/10.1016/j.cell.2019.04.038
Integrated supplementary information
Supplementary Figure 1 Control for DRB inhibition and release, as well as proportion of yeast spike-ins in TTchem-seq.
(a) Slot blot of RNA from HEK293 cells treated with 100 μM DRB for 3.5 hrs either without release, or with release for the indicated time period together with 10 min 1 mM 4SU labelling. (b) Bioanalyzer result of fragmented purified 4SU-RNA from cells treated with 100 μM DRB for 3.5 hrs either with or without release for the indicated time. All samples were labelled with 1 mM 4SU for 10 min prior to harvest. Gel like images of the electropherogram are shown on the top. (c) Percent of sequencing reads mapping to the yeast genome when using 1% total yeast RNA as spike-ins.
Supplementary Figure 2 Protocol optimization.
(a) Increasing treatment time of total RNA with sodium hydroxide (0.167 M) results in decreasing RNA fragment size. 1.2% denaturing TBE gel. (b) Bioanalyzer results comparing Qiagen minElute RNA clean-up of 4SU-RNA after streptavidin pull-out using the recommended protocol which selects for RNA > 200 nt or the modified protocol using an increased volume of ethanol.
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Supplementary Figs. 1 and 2.
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Gregersen, L.H., Mitter, R. & Svejstrup, J.Q. Using TTchem-seq for profiling nascent transcription and measuring transcript elongation. Nat Protoc 15, 604–627 (2020). https://doi.org/10.1038/s41596-019-0262-3
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DOI: https://doi.org/10.1038/s41596-019-0262-3
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