Li Xiao-Yong, Biggin Mark D
Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
Methods Mol Biol. 2012;809:3-26. doi: 10.1007/978-1-61779-376-9_1.
Immunoprecipitation of cross-linked chromatin in combination with microarrays (ChIP-chip) or ultra high-throughput sequencing (ChIP-seq) is widely used to map genome-wide in vivo transcription factor binding. Both methods employ initial steps of in vivo cross-linking, chromatin isolation, DNA fragmentation, and immunoprecipitation. For ChIP-chip, the immunoprecipitated DNA samples are then amplified, labeled, and hybridized to DNA microarrays. For ChIP-seq, the immunoprecipitated DNA is prepared for a sequencing library, and then the library DNA fragments are sequenced using ultra high-throughput sequencing platform. The protocols described here have been developed for ChIP-chip and ChIP-seq analysis of sequence-specific transcription factor binding in Drosophila embryos. A series of controls establish that these protocols have high sensitivity and reproducibility and provide a quantitative measure of relative transcription factor occupancy. The quantitative nature of the assay is important because regulatory transcription factors bind to highly overlapping sets of thousands of genomic regions and the unique regulatory specificity of each factor is determined by relative moderate differences in occupancy between factors at commonly bound regions.
交联染色质免疫沉淀结合微阵列（ChIP-chip）或超高通量测序（ChIP-seq）被广泛用于全基因组范围内绘制体内转录因子结合图谱。这两种方法都采用体内交联、染色质分离、DNA片段化和免疫沉淀的初始步骤。对于ChIP-chip，然后将免疫沉淀的DNA样本进行扩增、标记，并与DNA微阵列杂交。对于ChIP-seq，将免疫沉淀的DNA制备用于测序文库，然后使用超高通量测序平台对文库DNA片段进行测序。这里描述的方案是为果蝇胚胎中序列特异性转录因子结合的ChIP-chip和ChIP-seq分析而开发的。一系列对照表明，这些方案具有高灵敏度和可重复性，并提供了相对转录因子占据率的定量测量。该测定的定量性质很重要，因为调节性转录因子与数千个基因组区域的高度重叠集合结合，并且每个因子独特的调节特异性由共同结合区域中因子之间占据率的相对适度差异决定。
