Genome-wide measurement of protein-DNA binding dynamics using competition ChIP.
Genome-wide measurement of protein-DNA binding dynamics using competition ChIP.
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使用竞争 ChIP 对蛋白质-DNA 结合动力学进行全基因组测量。
DOI：
10.1038/nprot.2013.077
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发表时间：
2013
期刊：
影响因子：
14.8
通讯作者：
Lieb,JasonD
中科院分区：
文献类型：
--
作者：
Lickwar,ColinR;Mueller,Florian;Lieb,JasonD
竞争染色质免疫沉淀（竞争ChIP）使实验人员能够根据检测方法在单个位点或整个基因组中测量蛋白质-DNA动力学。竞争ChIP依赖于含有两个拷贝的单个DNA相关因子的细胞，每个拷贝的因子表位有差异标记。其中一个副本是组成性的，第二个副本是作为竞争者诱导的。与离散基因组位置相关的同种型比率通过ChIP芯片（ChIP芯片）或ChIP测序（ChIP序列）检测。蛋白质的驻留同种型在每个结合位置处被竞争物替换的速率使得能够计算该因子在基因组范围内的每个相互作用位点处的停留时间。在这里，我们提供了一个详细的协议，设计和进行竞争ChIP实验在酿酒酵母，这需要105天才能完成（不包括菌株生产和表征，这可能需要长达6个月）。该方案中包括下游生物信息学分析的指南，以提取整个基因组的停留时间。
Competition chromatin immunoprecipitation (competition ChIP) enables experimenters to measure protein-DNA dynamics at a single locus or across the entire genome, depending on the detection method. Competition ChIP relies on a cell containing two copies of a single DNA-associated factor, with each copy of the factor differentially epitope tagged. One of the copies is expressed constitutively and the second is induced as a competitor. The ratio of isoforms associated with discrete genomic locations is detected by ChIP-on-chip (ChIP-chip) or ChIP-sequencing (ChIP-seq). The rate at which the resident isoform of the protein is replaced by the competitor at each binding location enables the calculation of residence time for that factor at each site of interaction genome wide. Here we provide a detailed protocol for designing and performing competition ChIP experiments inSaccharomyces cerevisiae, which takes ∼5 d to complete (not including strain production and characterizations, which may take as long as 6 months). Included in this protocol are guidelines for downstream bioinformatic analysis to extract residence times throughout the genome.