Saini Amarjit, Sundberg Carl Johan
Department of Laboratory Medicine, Clinical Physiology Karolinska Institutet and Unit of Clinical Physiology, Karolinska University Hospital, 141 86, Stockholm, Sweden.
Molecular Exercise Physiology, Department of Physiology and Pharmacology, Karolinska Institutet, 171 77, Stockholm, Sweden.
Methods Mol Biol. 2018;1689:127-138. doi: 10.1007/978-1-4939-7380-4_11.
Chromatin immunoprecipitation (ChIP) is an invaluable method for studying interactions between histone proteins and genomic DNA regions and transcriptional regulation using antibodies to enrich genomic regions associated with these epitopes. Either to monitor the presence of histones with post-translational modifications at specific genomic locations or to measure transcription factor interactions with a candidate target gene, protein-DNA complexes are most commonly crosslinked using formaldehyde, which stabilizes these transient interactions. Chromatin is then fragmented to allow separation of genomic fragments bound by the histone or transcription factor of interest away from those that are unbound. Following immunoprecipitation, formaldehyde crosslinks are reversed and enriched DNA fragments are purified. While some investigators have successfully performed ChIP experiments from crosslinked skeletal muscle in cell culture, the process is relatively inefficient compared to whole tissue. This chapter provides protocols specifically designed for the crosslinking and immunoprecipitation of human skeletal muscle biopsy samples in preparation for chromatin immunoprecipitation-sequencing (ChIP-seq).
染色质免疫沉淀（ChIP）是一种非常有价值的方法，用于研究组蛋白与基因组DNA区域之间的相互作用以及转录调控，它使用抗体来富集与这些表位相关的基因组区域。无论是监测特定基因组位置上具有翻译后修饰的组蛋白的存在，还是测量转录因子与候选靶基因的相互作用，蛋白质-DNA复合物最常用甲醛进行交联，甲醛可稳定这些瞬时相互作用。然后将染色质片段化，以使与感兴趣的组蛋白或转录因子结合的基因组片段与未结合的片段分离。免疫沉淀后，逆转甲醛交联并纯化富集的DNA片段。虽然一些研究人员已成功在细胞培养中对交联的骨骼肌进行了ChIP实验，但与全组织相比，该过程效率相对较低。本章提供了专门设计用于人骨骼肌活检样本交联和免疫沉淀的方案，以准备进行染色质免疫沉淀测序（ChIP-seq）。
