Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.
Department of Chemistry and Biochemistry, SUNY Brockport, Brockport, NY 14420, USA.
STAR Protoc. 2021 Jun 15;2(2):100554. doi: 10.1016/j.xpro.2021.100554. eCollection 2021 Jun 18.
We describe a genome-wide DNA double-strand break (DSB) mapping technique, Break-seq. In this protocol, we provide step-by-step instructions for cell embedment in agarose, in-gel DSB labeling and subsequent capture, followed by standard Illumina library construction and sequencing. We also provide the framework for sequence data processing and DSB peak identification. Finally, we present a custom-designed 3D-printed device for processing agarose-embedded DNA samples. The protocol is applicable to , as well as mammalian suspension, adherent, and 3D organoid cell cultures. For complete details on the use and execution of this protocol, please refer to Hoffman et al. (2015) and Chakraborty et al. (2020).
我们描述了一种全基因组 DNA 双链断裂 (DSB) 作图技术，即 Break-seq。在本方案中，我们提供了在琼脂糖中包埋细胞、胶内 DSB 标记和随后的捕获、标准 Illumina 文库构建和测序的分步说明。我们还提供了序列数据处理和 DSB 峰识别的框架。最后，我们介绍了一种定制的 3D 打印设备，用于处理琼脂糖包埋的 DNA 样品。该方案适用于哺乳动物悬浮、贴壁和 3D 类器官细胞培养物。有关此方案的使用和执行的完整详细信息，请参阅 Hoffman 等人（2015 年）和 Chakraborty 等人（2020 年）。
