[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"detail-sidebar-cat-0-en-105":3,"doc-detail-447074-en":59,"doc-seo-447074-105":80},{"code":4,"msg":5,"data":6},0,"success",[7,13,18,23,28,33,38,43,48,51,55],{"id":8,"doc_module":4,"doc_module_name":9,"category_name":10,"show_sort_weight":11,"slug":12},1,"Document","Story & Novel",90,"story-novel",{"id":14,"doc_module":4,"doc_module_name":9,"category_name":15,"show_sort_weight":16,"slug":17},2,"Literature",80,"literature",{"id":19,"doc_module":4,"doc_module_name":9,"category_name":20,"show_sort_weight":21,"slug":22},4,"Exam",70,"exam",{"id":24,"doc_module":4,"doc_module_name":9,"category_name":25,"show_sort_weight":26,"slug":27},5,"Comic",60,"comic",{"id":29,"doc_module":4,"doc_module_name":9,"category_name":30,"show_sort_weight":31,"slug":32},6,"Technology",50,"technology",{"id":34,"doc_module":4,"doc_module_name":9,"category_name":35,"show_sort_weight":36,"slug":37},7,"Healthcare",40,"healthcare",{"id":39,"doc_module":4,"doc_module_name":9,"category_name":40,"show_sort_weight":41,"slug":42},8,"Research & Report",30,"research-report",{"id":44,"doc_module":4,"doc_module_name":9,"category_name":45,"show_sort_weight":46,"slug":47},9,"Religion & Spirituality",20,"religion-spirituality",{"id":46,"doc_module":4,"doc_module_name":9,"category_name":49,"show_sort_weight":46,"slug":50},"World Cup","world-cup",{"id":52,"doc_module":4,"doc_module_name":9,"category_name":53,"show_sort_weight":52,"slug":54},10,"Lifestyle","lifestyle",{"id":56,"doc_module":4,"doc_module_name":9,"category_name":57,"show_sort_weight":24,"slug":58},19,"General","general",{"code":4,"msg":5,"data":60},{"doc_id":61,"user_id":62,"nickname":63,"user_avatar":64,"doc_module":4,"category_id":39,"category_name":40,"doc_title":65,"doc_description":66,"doc_content":67,"file_id":68,"file_url":69,"file_type":70,"file_size":71,"view_count":8,"is_deleted":4,"is_public":8,"is_downloadable":8,"audit_status":8,"page_count":72,"language":73,"language_code":74,"site_id":75,"html_lang":74,"table_of_contents":76,"faqs":77,"seo_title":78,"seo_description":66,"update_tm":79,"read_time":36},447074,962090760408,"pop out","https://ap-avatar.wpscdn.com/davatar_085a072bc5b1113ac321206ff7593b45","Optimized ChIP-exo for mammalian cells and patterned sequencing flow cells - Research report - Protocol","Optimized ChIP-exo enables near basepair-resolution mapping of genome-wide protein–DNA interactions by combining chromatin immunoprecipitation with exonuclease digestion. Broad adoption has been limited by lengthy workflows, multiple custom reactions, quality-control gaps, and incompatibilities with modern Illumina patterned-flow-cell platforms. This study systematically optimizes and adapts ChIP-exo library construction for mammalian cells, introducing MO-ChIP-exo and validating it against published protocols across suspension and adherent lineages.","G3, 2026, 16(1), jkaf270  \n[https://doi.org/10.1093/g3journal/jkaf270](https://doi.org/10.1093/g3journal/jkaf270)  \nAdvance Access Publication Date: 7 November 2025  \nInvestigation  \nOptimized ChIP-exo for mammalian cells and patterned sequencing flow cells  \nDaniela Q. James  ,* Sohini Mukherjee, C. Caiden Cannon, Shaun Mahony  *  \nCenter for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, United States  \n*Corresponding authors: Daniela Q, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 411 South Frear Building, 150 Old Coaly Way, University Park, PA 16802, [USA. James. Email: daniela.james@psu.edu](USA. James. Email: daniela.james@psu.edu); Shaun Mahony, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 404 South Frear Building, 150 Old Coaly Way, University Park, PA 16802, [USA. Email: mahony@psu.edu](USA. Email: mahony@psu.edu)  \nBy combining chromatin immunoprecipitation (ChIP) with an exonuclease digestion of protein-bound DNA fragments, ChIP-exo characterizes genome-wide protein–DNA interactions at near basepair resolution. However, the widespread adoption of ChIP-exo has been hindered by several technical challenges, including lengthy protocols, the need for multiple custom reactions, and incompatibilities with recent Illumina sequencing platforms. To address these barriers, we systematically optimized and adapted the ChIP-exo library construction protocol for the unique requirements of mammalian cells and current sequencing technologies. We introduce a mammalian-optimized ChIP-exo (MO-ChIP-exo) protocol that builds upon previous ChIP-exo protocols with systematic optimization of crosslinking, harvesting, and library construction. We validate MO-ChIP-exo by comparing it with previously published ChIP-exo protocols and demonstrate its adaptability to both suspension (K562) and adherent (HepG2, mESC) cell lines. This improved protocol provides a more robust and efficient method for generating high-quality ChIP-exo libraries from mammalian cells.  \nKeywords: ChIP-exo; protocols; sonication; chromatin; chromatin immunoprecipitation; NGS; NextSeq 2000; CTCF  \nIntroduction  \nCharacterizing the genome-wide binding locations of transcription factors and other regulatory proteins is crucial for understanding complex cellular functions. Chromatin immunoprecipitation (ChIP) combined with high-throughput sequencing (ChIP-seq) (Albert et al. 2007 ; Johnson et al. 2007) emerged as the main method to identify genome-wide patterns of protein–DNA binding in a wide variety of species and cell types (Dunham et al. 2012 ; Moore et al. 2020) . While newer approaches such as CUT&RUN (Skene and Henikoff 2017) and CUT&Tag (Kaya-Okur et al. 2019) have provided valuable alternatives, they are most popularly applied to characterize histone modifications (Abbasova et al. 2025) . ChIP-seq remains the preferred method to study mammalian transcription factor binding. ChIP-exo greatly improved upon the resolution of ChIP-seq by incorporating an exonuclease to digest immunoprecipitated DNA fragments in a strand-specific direction until blocked by a crosslinked protein (Rhee and Pugh 2011 , 2012) . Asa result, ChIP-exo yields sharper protein-bound fragments and lower background than ChIP-seq, enabling the identification of binding events at near basepair resolution. In yeast, ChIP-exo was applied to study transcription factor binding (Rhee and Pugh 2011) and to characterize the organization of individual histones (Rhee et al. 2014) . Most notably, ChIP-exo was used to characterize the genome-wide architecture of all yeast chromatin-associated proteins in the Yeast Epigenome Project (Rossi et al. 2021) . It has also been applied to screen a large  \ncohort of monoclonal antibodies generated by the Protein Capture Reagents Program (Lai et al. 2021) .  \nDespite its advantages, the widespread ado","cbCaicQ01VL9Odis","https://ap.wps.com/l/cbCaicQ01VL9Odis","pdf",1555359,16,"English","en",105,"# Introduction\n## ChIP-exo method and rationale\n## Limitations of existing ChIP-exo protocols\n## Goal and approach of the optimized protocol","[{\"question\":\"What is ChIP-exo and what resolution does it achieve?\",\"answer\":\"ChIP-exo combines chromatin immunoprecipitation with exonuclease digestion of protein-bound DNA fragments. It produces sharper, lower-background fragments and enables identification of binding events at near basepair resolution.\"},{\"question\":\"Why has ChIP-exo adoption been difficult?\",\"answer\":\"Widespread use has been hindered by lengthy protocols, the need for multiple custom reactions, limited quality-control checkpoints, and higher costs, along with incompatibilities with newer Illumina sequencing platforms.\"},{\"question\":\"How does MO-ChIP-exo improve library construction for mammalian cells?\",\"answer\":\"MO-ChIP-exo systematically optimizes crosslinking, harvesting, and library construction to match mammalian requirements and current sequencing technologies. It is validated by comparison with previously published protocols and shown to work in both suspension and adherent cell lines.\"}]","Optimized ChIP-exo for mammalian cells and patterned sequencing flow cells - Research report - Protocol | PDF",1790718681,{"code":4,"msg":81,"data":82},"ok",{"site_id":75,"language":74,"slug":83,"title":65,"keywords":84,"description":66,"schema_data":85,"social_meta":139,"head_meta":141,"extra_data":143,"updated_unix":144},"optimized-chip-exo-for-mammalian-cells-and-patterned-sequencing-flow-cells-research-report-protocol","",{"@graph":86,"@context":138},[87,101,121],{"@type":88,"itemListElement":89},"BreadcrumbList",[90,94,96,99],{"item":91,"name":92,"@type":93,"position":8},"https://docshare.wps.com","Home","ListItem",{"item":95,"name":9,"@type":93,"position":14},"https://docshare.wps.com/document/",{"item":97,"name":40,"@type":93,"position":98},"https://docshare.wps.com/document/research-report/",3,{"item":100,"name":65,"@type":93,"position":19},"https://docshare.wps.com/document/optimized-chip-exo-for-mammalian-cells-and-patterned-sequencing-flow-cells-research-report-protocol/447074/",{"url":100,"name":65,"@type":102,"image":103,"author":108,"headline":65,"publisher":110,"fileFormat":113,"inLanguage":74,"description":66,"dateModified":114,"datePublished":115,"encodingFormat":113,"isAccessibleForFree":116,"interactionStatistic":117},"DigitalDocument",{"url":104,"@type":105,"width":106,"height":107},"https://docshare.wps.com/thumbnails/optimized-chip-exo-for-mammalian-cells-and-patterned-sequencing-flow-cells-research-report-protocol/447074.png","ImageObject",300,407,{"name":63,"@type":109},"Person",{"url":91,"name":111,"@type":112},"DocShare","Organization","application/pdf","2026-09-30","2026-09-29",true,{"@type":118,"interactionType":119,"userInteractionCount":8},"InteractionCounter",{"@type":120},"ViewAction",{"@type":122,"mainEntity":123},"FAQPage",[124,130,134],{"name":125,"@type":126,"acceptedAnswer":127},"What is ChIP-exo and what resolution does it achieve?","Question",{"text":128,"@type":129},"ChIP-exo combines chromatin immunoprecipitation with exonuclease digestion of protein-bound DNA fragments. It produces sharper, lower-background fragments and enables identification of binding events at near basepair resolution.","Answer",{"name":131,"@type":126,"acceptedAnswer":132},"Why has ChIP-exo adoption been difficult?",{"text":133,"@type":129},"Widespread use has been hindered by lengthy protocols, the need for multiple custom reactions, limited quality-control checkpoints, and higher costs, along with incompatibilities with newer Illumina sequencing platforms.",{"name":135,"@type":126,"acceptedAnswer":136},"How does MO-ChIP-exo improve library construction for mammalian cells?",{"text":137,"@type":129},"MO-ChIP-exo systematically optimizes crosslinking, harvesting, and library construction to match mammalian requirements and current sequencing technologies. It is validated by comparison with previously published protocols and shown to work in both suspension and adherent cell lines.","https://schema.org",{"og:url":100,"og:type":140,"og:title":65,"og:site_name":111,"og:description":66},"article",{"robots":142,"canonical":100},"index,follow",{"doc_id":61,"site_id":75},1790792614]