Transcription Factor Chromatin Immunoprecipitation in Endothelial Cells

Interactions between DNA and proteins are crucial for the regulation of gene expression. Chromatin immunoprecipitation (ChIP) is a powerful technique that allows the study of specific protein–DNA interactions in cultured cells and fresh or fixed tissue. Chromatin is isolated and sheared, and antibodies against the protein(s) of interest are used to isolate specific protein–DNA complexes. Subsequent analysis by real-time polymerase chain reaction (qPCR) or next-generation sequencing (NGS) allows identification and quantification of the co-purified DNA fragments, and NGS also gives insight into the genomic binding sites of a protein. Here we describe a cross-linking ChIP (X-ChIP) protocol, based around the example of a myc-tagged Proline-Rich Homeodomain (PRH) protein expressed in human umbilical vein endothelial cells. We also describe how to analyse specific known or suspected binding sites using quantitative PCR as well as how to analyse genome-wide binding from ChIP sequencing data.