Chromatin immunoprecipitation with mouse adipocytes using hypotonic buffer to enrich nuclear fraction before fixation

Abstract

Summary: Chromatin immunoprecipitation (ChIP) experiments with differentiated adipocytes are challenging because lipid droplets interfere with immunoprecipitation efficiency. Here, the author describes optimized procedures to minimize the burden of lipid droplets by using hypotonic buffer to enrich nuclear fraction before formaldehyde crosslinking, thus increasing the sensitivity and specificity of ChIP experiments with differentiated adipocytes. The author also describes steps for after fixation, including sonication, immunoprecipitation, washing, reverse-crosslinking, and purification. This protocol is compatible with ChIP-qPCR and ChIP-seq of various transcription factors and histone modifications.For complete details on the use and execution of this protocol, please refer to Hiraike et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Metabolism
• Chromatin Immunoprecipitation (ChIP)
• Cell Differentiation