Laboratory of Molecular Biology, Wageningen University, Wageningen, The Netherlands.
Nat Protoc. 2010 Mar;5(3):457-72. doi: 10.1038/nprot.2009.244. Epub 2010 Feb 18.
Chromatin immunoprecipitation (ChIP) is a powerful technique to study interactions between transcription factors (TFs) and DNA in vivo. For genome-wide de novo discovery of TF-binding sites, the DNA that is obtained in ChIP experiments needs to be processed for sequence identification. The sequences can be identified by direct sequencing (ChIP-SEQ) or hybridization to microarrays (ChIP-CHIP). Given the small amounts of DNA that are usually obtained in ChIP experiments, successful and reproducible sample processing is challenging. Here we provide a detailed procedure for ChIP of plant TFs, as well as protocols for sample preparation for ChIP-SEQ and for ChIP-CHIP. Our ChIP procedure is optimized for high signal-to-noise ratio starting with tissue fixation, followed by nuclei isolation, immunoprecipitation, DNA amplification and purification. We also provide a guide for primary data analysis of ChIP-SEQ data. The complete protocol for ChIP-SEQ/ChIP-CHIP sample preparation starting from plant harvest takes approximately 7 d.
染色质免疫沉淀（ChIP）是研究转录因子（TFs）与体内 DNA 相互作用的强大技术。对于全基因组的 TF 结合位点的从头发现，ChIP 实验中获得的 DNA 需要进行序列鉴定。这些序列可以通过直接测序（ChIP-SEQ）或与微阵列杂交（ChIP-CHIP）来识别。鉴于 ChIP 实验中通常获得的 DNA 量较少，因此成功且可重复的样品处理具有挑战性。在这里，我们提供了一种植物 TF 的 ChIP 的详细程序，以及用于 ChIP-SEQ 和 ChIP-CHIP 的样品制备的方案。我们的 ChIP 程序从组织固定开始，经过核分离、免疫沉淀、DNA 扩增和纯化，优化了高信噪比。我们还为 ChIP-SEQ 数据的初步数据分析提供了指导。从植物收获开始，进行完整的 ChIP-SEQ/ChIP-CHIP 样品制备大约需要 7 天。
