Building a robust chromatin immunoprecipitation (ChIP) method with substantially improved efficiency

Abstract

Chromatin immunoprecipitation (ChIP) is the gold-standard method to detect the interactions between proteins and chromatin, and is a powerful tool to identify epigenetic modifications. Although ChIP protocols for plant species have been developed, many specific features of plants, especially woody plants, still hinder the efficiency of immunoprecipitation, resulting inefficient ChIP enrichment. There is an active demand for a highly efficient ChIP protocol. In the present study, we employed Betula platyphylla (birch) and Arabidopsis thaliana as the research materials, and five factors closely associated with ChIP efficiency were identified, including crosslinking, chromatin concentration using centrifugal filter, using new immunoprecipitation buffer, rescue DNA with proteinase K, and using sucrose to increase immunoprecipitation efficiency. Optimization of any these factors can significantly improve ChIP efficiency. Considering these factors together, a robust ChIP protocol was developed, for which the average fold enrichments were 16.88 and 6.43 fold of that gained using standard ChIP in birch and Arabidopsis, respectively. As this built ChIP method works well in both birch and Arabidopsis, it should be also suitable for other woody and herbaceous species. In addition, this ChIP method make it is possible to detect low-abundance TF-DNA interactions, and may extend the application of ChIP in plant kingdom. One sentence summaryBuilding a ChIP method that increases fold enrichment of birch by 16 folds in average and is adapted for both woody and herbaceous plants.