Abstract

Chromatin immunoprecipitation (ChIP) is a powerful tool for the characterization of covalent histone modifications and DNA–histone interactions in vivo. The procedure includes DNA–histone cross-linking in chromatin, shearing DNA into smaller fragments, immunoprecipitation with antibodies against the histone modifications of interest, followed by PCR identification of associated DNA sequences. In this protocol, we describe a simplified and optimized version of ChIP assay by reducing the number of experimental steps and isolation solutions and shortening preparation times. We include a nuclear isolation step before chromatin shearing, which provides a good yield of high-quality DNA resulting in at least 15 |[mu]|g of DNA from each immunoprecipitated sample (from 0.2 to 0.4 g of starting tissue material) sufficient to test |[ge]|25 genes of interest. This simpler and cost-efficient protocol has been applied for histone-modification studies of various Arabidopsis thaliana tissues and is easy to adapt for other systems as well.