Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-CHIP)
Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-CHIP)
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DOI：
10.1038/nprot.2009.244
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发表时间：
2010-01-01
期刊：
影响因子：
14.8
通讯作者：
Angenent, Gerco C.
中科院分区：
文献类型：
--
作者：
Kaufmann, Kerstin;Muino, Jose M.;Angenent, Gerco C.
染色质免疫沉淀(ChIP)是研究体内转录因子与DNA相互作用的一种强有力的技术。对于全基因组范围的TF结合位点的从头发现，在芯片实验中获得的DNA需要进行序列鉴定。序列可通过直接测序(CHIP-SEQ)或与微阵列杂交(CHIP-CHIP)进行鉴定。考虑到通常在芯片实验中获得的少量DNA，成功和可重复性的样本处理是具有挑战性的。在这里，我们提供了植物TF芯片的详细程序，以及CHIP-SEQ和CHIP-CHIP样品制备方案。我们的芯片程序针对高信噪比进行了优化，从组织固定开始，然后是核分离、免疫沉淀、DNA扩增和纯化。我们还为芯片序列数据的原始数据分析提供了指南。从植物收获开始，CHIP-SEQ/CHIP-CHIP样品制备的完整流程大约需要7天。
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 similar to 7 d.