Protocol for chromatin immunoprecipitation of chromatin-binding proteins in Schizosaccharomyces pombe using a dual-crosslinking approach.

IF 1.3 Q4 BIOCHEMICAL RESEARCH METHODS STAR Protocols Pub Date : 2025-03-13 DOI:10.1016/j.xpro.2025.103695
Jasbeer S Khanduja, Mo Motamedi
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Abstract

Single-crosslink chromatin immunoprecipitation (ChIP) is often ineffective at mapping the binding sites of chromatin-binding proteins that indirectly interact with DNA. Here, we present a protocol to map the genomic occupancy of different chromatin regulators and an RNA exosome adapter subunit in Schizosaccharomyces pombe using dual-crosslinking ChIP. We describe steps for cell growth, dual-crosslinking, cell lysis, sonification, and immunoprecipitation. We then detail procedures for washing, crosslink reversal, and DNA purification for downstream analysis using ChIP-qPCR and ChIP sequencing. For complete details on the use and execution of this protocol, please refer to Khanduja et al.1.

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单交联染色质免疫沉淀(ChIP)通常不能有效地绘制与 DNA 间接相互作用的染色质结合蛋白的结合位点。在这里,我们介绍了一种利用双交联 ChIP 测绘不同染色质调节因子和 RNA 外泌体适配亚基在小鼠中的基因组占位情况的方法。我们描述了细胞生长、双交联、细胞裂解、超声和免疫沉淀的步骤。然后,我们详细介绍了清洗、交联反转和 DNA 纯化的步骤,以便使用 ChIP-qPCR 和 ChIP 测序进行下游分析。有关本方案使用和执行的完整细节,请参阅 Khanduja 等人的文章1。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
STAR Protocols
STAR Protocols Biochemistry, Genetics and Molecular Biology-General Biochemistry, Genetics and Molecular Biology
CiteScore
2.00
自引率
0.00%
发文量
789
审稿时长
10 weeks
期刊介绍:
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