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High-resolution, ultrasensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS
- Source :
- Nat Protoc
- Publication Year :
- 2020
- Publisher :
- Springer Science and Business Media LLC, 2020.
-
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).
- Subjects :
- DNA Replication
Genome instability
Streptavidin
DNA Repair
DNA damage
genetic processes
Cell
Genomics
Computational biology
Genome
Genomic Instability
Article
General Biochemistry, Genetics and Molecular Biology
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
medicine
Humans
DNA Breaks, Double-Stranded
030304 developmental biology
0303 health sciences
Primed In Situ Labeling
High-Throughput Nucleotide Sequencing
DNA
Meiosis
Eukaryotic Cells
medicine.anatomical_structure
chemistry
Biotinylation
030217 neurology & neurosurgery
Subjects
Details
- ISSN :
- 17502799 and 17542189
- Volume :
- 16
- Database :
- OpenAIRE
- Journal :
- Nature Protocols
- Accession number :
- edsair.doi.dedup.....f4c622daa4ff5eb6fe6b0ca7d698bc3c