Department of Biology, Carolina Center for the Genome Sciences, Curriculum in Genetics and Molecular Biology and Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina, USA.
Nat Protoc. 2013;8(7):1337-53. doi: 10.1038/nprot.2013.077. Epub 2013 Jun 13.
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 in Saccharomyces 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.
竞争染色质免疫沉淀（competition ChIP）使实验者能够根据检测方法，在单个基因座或整个基因组范围内测量蛋白质-DNA 动态。竞争 ChIP 依赖于一个细胞中含有两个单一位点相关因子的拷贝，每个因子的拷贝都有不同的表位标记。一个拷贝是组成型表达的，第二个拷贝是作为竞争物诱导的。通过 ChIP-on-chip（ChIP-chip）或 ChIP-sequencing（ChIP-seq）检测与离散基因组位置相关的异构体的比例。通过竞争在每个结合位置替换驻留异构体的速度，可计算该因子在整个相互作用基因组上每个位置的停留时间。本文提供了在酿酒酵母中设计和进行竞争 ChIP 实验的详细方案，该方案大约需要 5 天（不包括菌株的生产和特征分析，这可能需要长达 6 个月的时间）。该方案包括提取整个基因组中停留时间的下游生物信息学分析指南。
