Deliard Sandra, Zhao Jianhua, Xia Qianghua, Grant Struan F A
Division of Human Genetics, Children's Hospital of Philadelphia Research Institute, USA.
J Vis Exp. 2013 Apr 19(74):50286. doi: 10.3791/50286.
ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions. For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers. Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.
染色质免疫沉淀测序（ChIP-seq）方法直接提供全基因组覆盖，其中将染色质免疫沉淀（ChIP）与大规模平行测序相结合，可用于识别体内与转录因子结合的哺乳动物DNA序列库。“下一代”基因组测序技术相比旧技术，能以成本效益方式产生的序列量增加1至2个数量级，从而使ChIP-seq方法能够直接提供全基因组覆盖，以有效分析哺乳动物蛋白质-DNA相互作用。对于成功的ChIP-seq方法，必须生成高质量的ChIP DNA模板以获得最佳测序结果。本描述基于对2型糖尿病发病机制中最密切相关基因（即转录因子7样2（TCF7L2））的蛋白质产物的经验。该因子也与多种癌症有关。概述了如何从结肠癌细胞系HCT116生成高质量的ChIP DNA模板，以便通过测序构建高分辨率图谱，确定TCF7L2结合的基因，从而进一步深入了解其在复杂性状发病机制中的关键作用。
