Department of Genetics, Medical Research Council (MRC), University of Cambridge, Cambridge, UK.
Methods Mol Biol. 2023;2693:61-71. doi: 10.1007/978-1-0716-3342-7_5.
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a widely used technique for genome-wide mapping of protein-DNA interactions and epigenetic marks in vivo. Recent studies have suggested an important role of heat shock protein 90 (Hsp90) in chromatin. This molecular chaperone assists other proteins to acquire their mature and functional conformation and helps in the assembly of many complexes. In this chapter, we provide specific details on how to perform Hsp90 ChIP-seq from Drosophila Schneider (S2) cells. Briefly, cells are simultaneously lyzed and reversibly cross-linked to stabilize protein-DNA interactions. Chromatin is prepared from isolated nuclei and sheared by sonication. Hsp90-bound loci are immunoprecipitated and the corresponding DNA fragments are purified and sequenced. The described approach revealed that Hsp90 binds close to the transcriptional start site of around one-third of all Drosophila coding genes and characterized the role of the chaperone at chromatin.
染色质免疫沉淀测序（ChIP-seq）是一种广泛用于在体内对蛋白质-DNA 相互作用和表观遗传标记进行全基因组作图的技术。最近的研究表明热休克蛋白 90（Hsp90）在染色质中起着重要作用。这种分子伴侣协助其他蛋白质获得其成熟和功能构象，并有助于许多复合物的组装。在本章中，我们提供了有关如何从果蝇 Schneider（S2）细胞中进行 Hsp90 ChIP-seq 的具体细节。简而言之，细胞同时裂解并可逆交联以稳定蛋白质-DNA 相互作用。从分离的核中制备染色质，并通过超声处理进行剪切。Hsp90 结合的基因座被免疫沉淀，相应的 DNA 片段被纯化和测序。所描述的方法表明，Hsp90 结合在大约三分之一的所有果蝇编码基因的转录起始位点附近，并表征了伴侣在染色质中的作用。
