Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
Howard Hughes Medical Institute, Seattle, Washington, USA.
Nat Protoc. 2018 May;13(5):1006-1019. doi: 10.1038/nprot.2018.015. Epub 2018 Apr 12.
Cleavage under targets and release using nuclease (CUT&RUN) is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. As only the targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has exceptionally low background levels. CUT&RUN outperforms the most widely used chromatin immunoprecipitation (ChIP) protocols in resolution, signal-to-noise ratio and depth of sequencing required. In contrast to ChIP, CUT&RUN is free of solubility and DNA accessibility artifacts and has been used to profile insoluble chromatin and to detect long-range 3D contacts without cross-linking. Here, we present an improved CUT&RUN protocol that does not require isolation of nuclei and provides high-quality data when starting with only 100 cells for a histone modification and 1,000 cells for a transcription factor. From cells to purified DNA, CUT&RUN requires less than a day at the laboratory bench and requires no specialized skills.
在靶标处切割并使用核酸酶释放（CUT&RUN）是一种表观基因组分析策略，其中微球菌核酸酶靶向抗体控制切割将特定的蛋白-DNA 复合物释放到上清液中，用于进行配对末端 DNA 测序。由于只有靶向片段进入溶液，而绝大多数 DNA 留在后面，因此 CUT&RUN 的背景水平非常低。CUT&RUN 在分辨率、信号与噪声比以及所需测序深度方面优于最广泛使用的染色质免疫沉淀（ChIP）方案。与 ChIP 不同，CUT&RUN 没有可溶性和 DNA 可及性伪影，并且已被用于分析不溶性染色质，并在不交联的情况下检测长程 3D 接触。在这里，我们提出了一种改进的 CUT&RUN 方案，它不需要分离细胞核，并且在起始时仅用 100 个细胞进行组蛋白修饰和 1000 个细胞进行转录因子分析时，也能提供高质量的数据。从细胞到纯化的 DNA，CUT&RUN 在实验室工作台上不到一天的时间内完成，并且不需要专门的技能。
