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High-resolution, ultrasensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS

Abstract

DNA double-strand breaks (DSBs) are implicated in various physiological processes, such as class-switch recombination or crossing-over during meiosis, but also present a threat to genome stability. Extensive evidence shows that DSBs are a primary source of chromosome translocations or deletions, making them a major cause of genomic instability, a driving force of many diseases of civilization, such as cancer. Therefore, there is a great need for a precise, sensitive, and universal method for DSB detection, to enable both the study of their mechanisms of formation and repair as well as to explore their therapeutic potential. We provide a detailed protocol for our recently developed ultrasensitive and genome-wide DSB detection method: immobilized direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (i-BLESS), which relies on the encapsulation of cells in agarose beads and labeling breaks directly and specifically with biotinylated linkers. i-BLESS labels DSBs with single-nucleotide resolution, allows detection of ultrarare breaks, takes 5 d to complete, and can be applied to samples from any organism, as long as a sufficient amount of starting material can be obtained. We also describe how to combine i-BLESS with our qDSB-Seq approach to enable the measurement of absolute DSB frequencies per cell and their precise genomic coordinates at the same time. Such normalization using qDSB-Seq is especially useful for the evaluation of spontaneous DSB levels and the estimation of DNA damage induced rather uniformly in the genome (e.g., by irradiation or radiomimetic chemotherapeutics).

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Fig. 1: Overview of the i-BLESS method.
Fig. 2: Encapsulation of cells in agarose beads.
Fig. 3: Spheroplasting and lysis.
Fig. 4: Anticipated results.

Data availability

The raw data underlying Fig. 4b–d are deposited to the NCBI Sequence Read Archive (SRA) with accession codes SRP125409 and SRP189465.

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Acknowledgements

We thank K. Jodkowska and T. Biernacki for help with the preparation of Supplementary Video 1. We also thank A. Kudlicki for critical reading of the manuscript. This work was supported by the Foundation for Polish Science (TEAM to K.G.) and the Polish National Science Centre (2015/17/D/NZ2/03711 to M.S.) and an NIH grant R01GM112131 to M.R.

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Authors

Contributions

K.G. supervised the study, A.B., M.S., P.P., M.R., and K.G. designed the experiments, A.B. and M.S. performed the experiments, Y.Z. and M.R. performed bioinformatic analysis, A.B., M.S., Y.Z., P.P., M.R., and K.G. analyzed the results, and A.B. and K.G. wrote the manuscript. All authors read and edited the manuscript.

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Correspondence to Krzysztof Ginalski.

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The authors declare no competing interests.

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

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Related links

Key references using this protocol

Biernacka, A. et al. Commun. Biol. 1, 181 (2018): https://doi.org/10.1038/s42003-018-0165-9

Zhu, Y. et al. Nat. Commun. 10, 2313 (2019): https://doi.org/10.1038/s41467-019-10332-8

Promonet, A. et al. Nat. Commun. 11, 3940 (2020): https://doi.org/10.1038/s41467-020-17858-2

Supplementary information

Reporting Summary

Supplementary Video 1

Encapsulation of human GM19239 cells in agarose beads.

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Biernacka, A., Skrzypczak, M., Zhu, Y. et al. High-resolution, ultrasensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS. Nat Protoc 16, 1034–1061 (2021). https://doi.org/10.1038/s41596-020-00448-3

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