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Genome-wide detection of DNA double-strand breaks by in-suspension BLISS

Abstract

sBLISS (in-suspension breaks labeling in situ and sequencing) is a versatile and widely applicable method for identification of endogenous and induced DNA double-strand breaks (DSBs) in any cell type that can be brought into suspension. sBLISS provides genome-wide profiles of the most consequential DNA lesion implicated in a variety of pathological, but also physiological, processes. In sBLISS, after in situ labeling, DSB ends are linearly amplified, followed by next-generation sequencing and DSB landscape analysis. Here, we present a step-by-step experimental protocol for sBLISS, as well as a basic computational analysis. The main advantages of sBLISS are (i) the suspension setup, which renders the protocol user-friendly and easily scalable; (ii) the possibility of adapting it to a high-throughput or single-cell workflow; and (iii) its flexibility and its applicability to virtually every cell type, including patient-derived cells, organoids, and isolated nuclei. The wet-lab protocol can be completed in 1.5 weeks and is suitable for researchers with intermediate expertise in molecular biology and genomics. For the computational analyses, basic-to-intermediate bioinformatics expertise is required.

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Fig. 1: Schematic depiction of the sBLISS workflow.
Fig. 2: Detailed overview of sBLISS library structure and read orientation.
Fig. 3: Experimental details for TK6 and enterocyte experiments.
Fig. 4: Genome browser views of mouse enterocyte sBLISS data.
Fig. 5: Analyses of mouse and human sBLISS data with the tutorial.
Fig. 6: Plotting the genome-wide density of DSB events.
Fig. 7: Integration of sBLISS data with mouse enterocyte gene expression levels.

Data availability

All raw sequencing data and processed mapped files used to generate Figs. 37 are made available through GEO: GSE145598. The TK6 data were generated as part of a published study (ref. 48), but the mapping pipeline has been updated since the original publication.

Code availability

All code is available on the BiCroLab GitHub repository. Code for the preprocessing and mapping pipeline can be found at https://github.com/BiCroLab/blissNP, and code for the analysis tutorials can be found at https://github.com/BiCroLab/blissNPanalysis.

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Acknowledgements

This work was supported by a Rubicon postdoctoral scholarship from the Dutch Research Council (NWO) to B.A.M.B.; by funding from the Ragnar Söderberg Foundation (Fellows in Medicine 2016) to M.B.; by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)–Project-ID 393547839–SFB 1361 and Project-ID 402733153-SPP 2202 to V.R.; and by funding from the Swedish Research Council (2018-02950), the Swedish Cancer Research Foundation (CAN 2018/728), the Ragnar Söderberg Foundation (Fellows in Medicine 2016), and the Strategic Research Programme in Cancer (StratCan) at Karolinska Institutet to N.C.

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Contributions

Conceptualization: B.A.M.B., N.C.; data curation: B.A.M.B., F.A., S.G., H.J.G.; formal analysis: F.A., S.G.; funding acquisition: B.A.M.B., S.I., M.B., V.R., N.C.; investigation: B.A.M.B., H.J.G., A.E.M.; methodology: B.A.M.B., N.C.; project administration: N.C.; software: F.A., S.G., G.P., S.S.; supervision: N.C.; visualization: B.A.M.B., F.A.; writing: B.A.M.B. with input from all the other authors.

Corresponding authors

Correspondence to Britta A. M. Bouwman or Nicola Crosetto.

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Competing interests

N.C. is a co-inventor in a US patent describing applications of BLISS for CRISPR off-target detection. The other authors declare no competing interests.

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Peer review information Nature Protocols thanks Toni Cathomen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Gothe, H. J. et al. Mol. Cell 75, 267–283.e12 (2019): https://doi.org/10.1016/j.molcel.2019.05.015

Dellino, G. I. et al. Nat. Genet. 51, 1011–1023 (2019): https://doi.org/10.1038/s41588-019-0421-z

Dziubańska-Kusibab, P. J. et al. Nat. Med. 26, 1063–1069 (2020): https://doi.org/10.1038/s41591-020-0908-2

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Bouwman, B.A.M., Agostini, F., Garnerone, S. et al. Genome-wide detection of DNA double-strand breaks by in-suspension BLISS. Nat Protoc 15, 3894–3941 (2020). https://doi.org/10.1038/s41596-020-0397-2

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