CUT&RUN Profiling of the Budding Yeast Epigenome.
CUT&RUN Profiling of the Budding Yeast Epigenome.
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DOI：
10.1007/978-1-0716-2257-5_9
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发表时间：
2022-01-01
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影响因子：
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通讯作者：
Henikoff, Steven
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文献类型：
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作者：
Brahma, Sandipan;Henikoff, Steven
表观基因组图谱是描述染色质景观与基于 DNA 的细胞过程（例如转录）控制之间关系的关键。使用核酸酶在目标下切割并释放 (CUT&RUN) 是一种原位染色质分析策略，其中通过抗体靶向微球菌核酸酶进行控制切割，溶解特定的蛋白质-DNA 复合物，用于双端 DNA 测序。当应用于芽殖酵母时，CUT&RUN 分析可生成组蛋白修饰、组蛋白变异、转录因子和 ATP 依赖性染色质重塑剂的精确全基因组图谱，同时避免与最常用的染色质分析技术染色质免疫沉淀 (ChIP) 相关的交联和溶解问题。此外，CUT&RUN 干净释放的靶向染色质复合物可用作后续天然免疫沉淀步骤 (CUT&RUN.ChIP) 的输入，以同时绘制全基因组单分子中的两个表位。 CUT&RUN 和 CUT&RUN.ChIP 本质上的低背景和高分辨率可以识别瞬时基因组特征，例如动态核小体重塑中间体。从细胞开始，进行CUT&RUN或CUT&RUN.ChIP，2天内即可获得纯化的DNA用于测序文库制备。
Mapping the epigenome is key to describe the relationship between chromatin landscapes and the control of DNA-based cellular processes such as transcription. Cleavage under targets and release using nuclease (CUT&RUN) is an in situ chromatin profiling strategy in which controlled cleavage by antibody-targeted Micrococcal Nuclease solubilizes specific protein-DNA complexes for paired-end DNA sequencing. When applied to budding yeast, CUT&RUN profiling yields precise genome-wide maps of histone modifications, histone variants, transcription factors, and ATP-dependent chromatin remodelers, while avoiding cross-linking and solubilization issues associated with the most commonly used chromatin profiling technique Chromatin Immunoprecipitation (ChIP). Furthermore, targeted chromatin complexes cleanly released by CUT&RUN can be used as input for a subsequent native immunoprecipitation step (CUT&RUN.ChIP) to simultaneously map two epitopes in single molecules genome-wide. The intrinsically low background and high resolution of CUT&RUN and CUT&RUN.ChIP allows for identification of transient genomic features such as dynamic nucleosome-remodeling intermediates. Starting from cells, one can perform CUT&RUN or CUT&RUN.ChIP and obtain purified DNA for sequencing library preparation in 2days.