An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites
An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites
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DOI：
10.7554/elife.21856
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发表时间：
2017-01-16
期刊：
影响因子：
7.7
通讯作者：
Henikoff, Steven
中科院分区：
文献类型：
--
作者：
Skene, Peter J.;Henikoff, Steven
我们描述了靶下切割和使用核酸酶释放（CUT& RUN），这是一种染色质分析策略，其中抗体靶向控制微球菌核酸酶的切割，将特定的蛋白质-DNA复合物释放到上清中，用于配对端DNA测序。与染色质免疫沉淀（ChIP）不同，它可以分解和溶解总染色质，而CUT& RUN是在原位进行的，允许定量高分辨率染色质制图和探测局部染色质环境。当应用于酵母和人类细胞核时，CUT& RUN产生了精确的转录因子谱，同时避免了交联和溶解问题。CUT& RUN操作简单，具有固有的鲁棒性，具有极低的背景，只需要与ChIP相似的1/10测序深度，使CUT& RUN在转录因子和染色质分析方面特别具有成本效益。当与本地ChIP-seq结合使用并应用于人类CTCF时，CUT& RUN以高分辨率绘制了定向远程接触位点。我们得出结论，CUT& RUN对蛋白质- dna相互作用的原位作图是ChIP-seq的一个有吸引力的替代方案。
We describe Cleavage Under Targets and Release Using Nuclease (CUT& RUN), a chromatin profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes total chromatin, CUT& RUN is performed in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, CUT& RUN yielded precise transcription factor profiles while avoiding crosslinking and solubilization issues. CUT& RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only similar to 1/10th the sequencing depth as ChIP, making CUT& RUN especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, CUT& RUN mapped directional long range contact sites at high resolution. We conclude that in situ mapping of protein-DNA interactions by CUT& RUN is an attractive alternative to ChIP-seq.