Chromatin Immunoprecipitation Followed by Next-Generation Sequencing in Ewing Sarcoma

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a widely used technique to study the distribution of transcription factors, histones, posttranslational modifications, or other DNA-associated proteins across the genome. This methodology is based on the isolation of DNA fragments bound by the protein of interest using specific antibodies. Computational analysis of sequencing libraries derived from the isolated DNA fragments will delineate genome-wide maps of protein-DNA interactions. ChIP-seq profiling of EWSR1::FLI1 oncogene has enormously contributed to the understanding of gene reprogramming in Ewing Sarcoma (EwS) defining EWSR1::FLI1 bound sites, the GGAA microsatellites, and the direct targets regulated by the translocation. ChIP-seq methodology has also provided novel biological insight into the proteins contributing to chromatin remodeling as well as the histone modification patterns associated with EWSR1::FLI1 genome-wide. This protocol provides detailed steps on how to conduct quantitative ChIP-seq experiments in EwS cell lines incorporating spike-in controls along with the core of a bioinformatic framework to analyze the results and infer novel biological knowledge.