{"title":"Protocol for chromatin immunoprecipitation of chromatin-binding proteins in Schizosaccharomyces pombe using a dual-crosslinking approach.","authors":"Jasbeer S Khanduja, Mo Motamedi","doi":"10.1016/j.xpro.2025.103695","DOIUrl":null,"url":null,"abstract":"<p><p>Single-crosslink chromatin immunoprecipitation (ChIP) is often ineffective at mapping the binding sites of chromatin-binding proteins that indirectly interact with DNA. Here, we present a protocol to map the genomic occupancy of different chromatin regulators and an RNA exosome adapter subunit in Schizosaccharomyces pombe using dual-crosslinking ChIP. We describe steps for cell growth, dual-crosslinking, cell lysis, sonification, and immunoprecipitation. We then detail procedures for washing, crosslink reversal, and DNA purification for downstream analysis using ChIP-qPCR and ChIP sequencing. For complete details on the use and execution of this protocol, please refer to Khanduja et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103695"},"PeriodicalIF":1.3000,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"STAR Protocols","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1016/j.xpro.2025.103695","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
Single-crosslink chromatin immunoprecipitation (ChIP) is often ineffective at mapping the binding sites of chromatin-binding proteins that indirectly interact with DNA. Here, we present a protocol to map the genomic occupancy of different chromatin regulators and an RNA exosome adapter subunit in Schizosaccharomyces pombe using dual-crosslinking ChIP. We describe steps for cell growth, dual-crosslinking, cell lysis, sonification, and immunoprecipitation. We then detail procedures for washing, crosslink reversal, and DNA purification for downstream analysis using ChIP-qPCR and ChIP sequencing. For complete details on the use and execution of this protocol, please refer to Khanduja et al.1.