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).

DNA 双链断裂 (DSB) 与多种生理过程有关，例如减数分裂期间的类别转换重组或交叉，但也对基因组稳定性构成威胁。大量证据表明，DSB 是染色体易位或缺失的主要来源，使其成为基因组不稳定的主要原因，而基因组不稳定是许多文明疾病（例如癌症）的驱动力。因此，非常需要一种精确、灵敏和通用的 DSB 检测方法，以便研究其形成和修复机制并探索其治疗潜力。我们为最近开发的超灵敏和全基因组 DSB 检测方法提供了详细的方案：固定化直接原位断裂标记、链霉亲和素富集和新一代测序 (i-BLESS)，该方法依赖于琼脂糖珠中的细胞封装和标记直接且特异地与生物素化接头断裂。i-BLESS 以单核苷酸分辨率标记 DSB，允许检测极罕见的断裂，需要 5 天才能完成，并且只要可以获得足够量的起始材料，就可以应用于任何生物体的样品。我们还描述了如何将 i-BLESS 与我们的 qDSB-Seq 方法相结合，以同时测量每个细胞的绝对 DSB 频率及其精确的基因组坐标。这种使用 qDSB-Seq 的标准化对于评估自发 DSB 水平和估计基因组中相当均匀诱导的 DNA 损伤（例如，通过辐射或拟放射化疗）特别有用。