Cotney Justin L, Noonan James P
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520.
Cold Spring Harb Protoc. 2015 Feb 2;2015(2):191-9. doi: 10.1101/pdb.prot084848.
Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) is a powerful method used to identify genome-wide binding patterns of transcription factors and distribution of various histone modifications associated with different chromatin states. In most published studies, ChIP-Seq has been performed on cultured cells grown under controlled conditions, allowing generation of large amounts of material in a homogeneous biological state. Although such studies have provided great insight into the dynamic landscapes of animal genomes, they do not allow the examination of transcription factor binding and chromatin states in adult tissues, developing embryonic structures, or tumors. Such knowledge is critical to understanding the information required to create and maintain a complex biological tissue and to identify noncoding regions of the genome directly involved in tissues affected by complex diseases such as autism. Studying these tissue types with ChIP-Seq can be challenging due to the limited availability of tissues and the lack of complex biological states able to be achieved in culture. These inherent differences require alterations of standard cross-linking and chromatin extraction typically used in cell culture. Here we describe a general approach for using small amounts of animal tissue to perform ChIP-Seq directed at histone modifications and transcription factors. Tissue is homogenized before treatment with formaldehyde to ensure proper cross-linking, and a two-step nuclear isolation is performed to increase extraction of soluble chromatin. Small amounts of soluble chromatin are then used for immunoprecipitation (IP) and prepared for multiplexed high-throughput sequencing.
染色质免疫沉淀结合高通量测序（ChIP-Seq）是一种强大的方法，用于识别全基因组范围内转录因子的结合模式以及与不同染色质状态相关的各种组蛋白修饰的分布。在大多数已发表的研究中，ChIP-Seq是在受控条件下培养的细胞上进行的，这使得能够在均匀的生物学状态下产生大量材料。尽管此类研究为动物基因组的动态景观提供了深刻见解，但它们无法检测成年组织、发育中的胚胎结构或肿瘤中的转录因子结合和染色质状态。这些知识对于理解创建和维持复杂生物组织所需的信息以及识别直接参与受自闭症等复杂疾病影响的组织的基因组非编码区域至关重要。由于组织可用性有限以及在培养中无法实现复杂的生物学状态，用ChIP-Seq研究这些组织类型可能具有挑战性。这些内在差异需要改变细胞培养中通常使用的标准交联和染色质提取方法。在这里，我们描述了一种使用少量动物组织进行针对组蛋白修饰和转录因子的ChIP-Seq的通用方法。在用甲醛处理之前将组织匀浆以确保适当交联，并进行两步核分离以增加可溶性染色质的提取。然后将少量可溶性染色质用于免疫沉淀（IP）并准备进行多重高通量测序。
