title: A chromatin immunoprecipitation (ChIP) protocol for use in whole human adipose tissue.
abstract: Chromatin immunoprecipitation (ChIP) has become a central method when studying in vivo protein-DNA interactions, with the major challenge being the hope to capture "authentic" interactions. While ChIP protocols have been optimized for use with specific cell types and tissues including adipose tissue-derived cells, a working ChIP protocol addressing the challenges imposed by fresh whole human adipose tissue has not been described. Utilizing human paired omental and subcutaneous adipose tissue obtained during elective abdominal surgeries, we have carefully identified and optimized individual steps in the ChIP protocol employed directly on fresh tissue fragments. We describe a complete working protocol for using ChIP on whole adipose tissue fragments. Specific steps required adaptation of the ChIP protocol to human whole adipose tissue. In particular, a cross-linking step was performed directly on fresh small tissue fragments. Nuclei were isolated before releasing chromatin, allowing better management of fat content; a sonication protocol to obtain fragmented chromatin was optimized. We also demonstrate the high sensitivity of immunoprecipitated chromatin from adipose tissue to freezing. In conclusion, we describe the development of a ChIP protocol optimized for use in studying whole human adipose tissue, providing solutions for the unique challenges imposed by this tissue. Unraveling protein-DNA interaction in whole human adipose tissue will likely contribute to elucidating molecular pathways contributing to common human diseases such as obesity and type 2 diabetes.
authors.affiliation: Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel;
authors.forename: Yulia
authors.initials: Y
authors.lastname: Haim
authors.forename: Tanya
authors.initials: T
authors.lastname: Tarnovscki
authors.forename: Dana
authors.initials: D
authors.lastname: Bashari
authors.forename: Assaf
authors.initials: A
authors.lastname: Rudich
mesh_terms: D000273:Adipose Tissue / Q000737:chemistry*; D001706:Biopsy; D002467:Cell Nucleus / Q000737:chemistry; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers; D006801:Humans; D007202:Indicators and Reagents; D016133:Polymerase Chain Reaction; D014021:Tissue Preservation; D014465:Ultrasonics
chemical_list: D003432:Cross-Linking Reagents; D017931:DNA Primers; D007202:Indicators and Reagents; D004247:DNA
keywords: ChIP; end-point PCR; protein-DNA interaction; transcriptional regulation; whole human adipose tissue