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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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.
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.
Peer review information Nature Protocols thanks Toni Cathomen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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