Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP.
Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP.
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DOI：
10.3791/53478
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发表时间：
2016-01-20
期刊：
影响因子：
--
通讯作者：
Fujii H
中科院分区：
文献类型：
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作者：
Fujita T;Fujii H
需要鉴定与特定基因组感兴趣区域相关的分子，以了解这些区域功能的调节机制。为了能够无偏见地识别与感兴趣的特定基因组区域相互作用的分子，我们最近开发了工程 DNA 结合分子介导的染色质免疫沉淀 (enChIP) 技术。在这里，我们描述如何使用 enChIP 分离特定的基因组区域并鉴定相关的蛋白质和 RNA。首先，用类转录激活子 (TAL) 蛋白或由催化失活形式的 Cas9 和引导 RNA 组成的成簇规则间隔短回文重复序列 (CRISPR) 复合物标记感兴趣的基因组区域。随后，染色质通过超声处理交联并断裂。然后对标记的位点进行免疫沉淀并逆转交联。最后，与分离的染色质相关的蛋白质或 RNA 分别进行质谱或 RNA 测序分析。这种方法可以成功鉴定与感兴趣的基因组区域相关的蛋白质和 RNA。
The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.