ChIP-Seq to analyze the binding of replication proteins to chromatin

Abstract

Chromatin immunoprecipitation (ChIP) is a widely used method to study interactions between proteins and discrete chromosomal loci in vivo. ChIP was originally developed for in vivo analysis of protein associations with candidate DNA sequences known or suspected to bind the protein of interest. The advent of DNA microarrays enabled the unbiased, genome-scale identification of all DNA sequences enriched by ChIP, providing a genomic map of a protein’s chromatin binding. This method, termed ChIP-chip, is broadly applicable and has been particularly valuable in DNA replication studies to map potential replication origins in Saccharomyces cerevisiae and other organisms based on the specific association of certain replication proteins with these chromosomal elements, which are distributed throughout the genome. More recently, high-throughput sequencing (HTS) technologies have replaced microarrays as the preferred method for genomic analysis of ChIP experiments, and this combination is termed ChIP-Seq. We present a detailed ChIP-Seq protocol for S. cerevisiae that can be adapted for different HTS platforms and for different organisms. We also outline general schemes for data analysis; however, HTS data analyses usually must be tailored specifically for individual studies, depending on the experimental design, data characteristics, and the genome being analyzed.