脂肪组织和脂肪细胞中的染色质免疫沉淀：如何进行和优化转录因子 DNA 结合的实验方案。

Chromatin Immunoprecipitation in Adipose Tissue and Adipocytes: How to Proceed and Optimize the Protocol for Transcription Factor DNA Binding.

机构信息

Institute for Diabetes and Cancer, Helmholtz Zentrum Muenchen, Neuherberg, Germany.

Institute for Diabetes and Endocrinology, Helmholtz Zentrum Muenchen, Neuherberg, Germany.

出版信息

Methods Mol Biol. 2024;2846:35-45. doi: 10.1007/978-1-0716-4071-5_3.

DOI:10.1007/978-1-0716-4071-5_3

PMID:39141228

Abstract

Chromatin immunoprecipitation (ChIP) coupled to qPCR or sequencing is a crucial experiment to determine direct transcriptional regulation under the control of specific transcriptional factors or co-regulators at loci-specific or pan-genomic levels.Here we provide a reliable method for processing ChIP from adipocytes or frozen adipose tissue collection, isolation of nuclei, cross-linking of protein-DNA complexes, chromatin shearing, immunoprecipitation, and DNA purification. We also discuss critical steps for optimizing the experiment to perform a successful ChIP in lipid-rich cells/tissues.

摘要

染色质免疫沉淀 (ChIP) 联合 qPCR 或测序是一种至关重要的实验方法，可用于在特定转录因子或共调节因子的控制下，在特定基因座或全基因组水平上确定直接转录调控。在这里，我们提供了一种从脂肪细胞或冷冻脂肪组织中提取 ChIP 的可靠方法，包括核分离、蛋白-DNA 复合物交联、染色质剪切、免疫沉淀和 DNA 纯化。我们还讨论了优化实验的关键步骤，以在富含脂质的细胞/组织中成功进行 ChIP。