High-resolution, ultrasensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS.
High-resolution, ultrasensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS.
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使用i-BLESS在真核细胞中进行高分辨率、超灵敏和定量的DNA双链断裂标记。
DOI：
10.1038/s41596-020-00448-3
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发表时间：
2021-03
期刊：
影响因子：
14.8
通讯作者：
Ginalski K
中科院分区：
文献类型：
--
作者：
Biernacka A;Skrzypczak M;Zhu Y;Pasero P;Rowicka M;Ginalski K
DNA双链断裂（DSB）涉及各种生理过程，如减数分裂过程中的类别转换重组或交换，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，是许多文明疾病（如癌症）的驱动力。因此，迫切需要一种精确、灵敏和通用的DSB检测方法，以研究其形成和修复机制并探索其治疗潜力。在这里，我们为我们最近开发的超灵敏和全基因组DSB检测方法i-BLESS提供了详细的方案，该方法依赖于琼脂糖珠中的细胞包封，并直接特异性地用生物素化接头标记断裂。i-BLESS标记具有单核苷酸分辨率的DSB，允许检测超罕见的断裂，需要五天才能完成，并且可以应用于任何生物体的样品，只要可以获得足够量的起始材料。我们还描述了如何将联合收割机i-BLESS与我们的qDSB-Seq方法相结合，从而能够同时测量每个细胞的绝对DSB频率及其精确的基因组坐标。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地诱导的DNA损伤（例如，由辐射或拟放射性化学治疗剂引起）特别有用。该方案描述了一种全基因组方法，用于超灵敏和定量检测DNA双链断裂（DSB），该方法依赖于将细胞包封在琼脂糖珠中并用生物素化接头标记断裂。
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 civilization diseases, such as cancer. Therefore, there is a great need for a precise, sensitive and universal method for DSB detection, to enable both studying mechanisms of their formation and repair as well as exploring their therapeutic potential. Here, we provide a detailed protocol for our recently developed ultra-sensitive and genome-wide DSB detection method i-BLESS, which relies on 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 ultra-rare breaks, takes five days to complete and can be applied to samples from any organism, as long as sufficient amount of starting material can be obtained. We also describe how to combine i-BLESS with our qDSB-Seq approach enabling measuring absolute DSB frequencies per cell and their precise genomic coordinates at the same time. Normalization with qDSB-Seq is especially useful for evaluation of spontaneous DSB levels and estimations of DNA damage induced rather uniformly in the genome (e.g. caused by irradiation or radiomimetic chemotherapeutics). This protocol describes a genome-wide approach for ultrasensitive and quantitative detection of DNA double-strand breaks (DSBs) relying on encapsulating cells in agarose beads and labeling breaks with biotinylated adapters.
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影响因子：
4.5
作者：
Chung WH;Zhu Z;Papusha A;Malkova A;Ira G
通讯作者：
Ira G
影响因子：
5.9
作者：
Biernacka A;Zhu Y;Skrzypczak M;Forey R;Pardo B;Grzelak M;Nde J;Mitra A;Kudlicki A;Crosetto N;Pasero P;Rowicka M;Ginalski K
通讯作者：
Ginalski K
Talen介导的诱变和基因组编辑。
DOI：
10.1007/978-1-4939-3771-4_2
发表时间：
2016
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Ma AC;Chen Y;Blackburn PR;Ekker SC
通讯作者：
Ekker SC
影响因子：
14.8
作者：
Hu J;Meyers RM;Dong J;Panchakshari RA;Alt FW;Frock RL
通讯作者：
Frock RL
影响因子：
48
作者：
Crosetto, Nicola;Mitra, Abhishek;Silva, Maria Joao;Bienko, Magda;Dojer, Norbert;Wang, Qi;Karaca, Elif;Chiarle, Roberto;Skrzypczak, Magdalena;Ginalski, Krzysztof;Pasero, Philippe;Rowicka, Maga;Dikic, Ivan
通讯作者：
Dikic, Ivan