Using ChIP-seq technology to generate high-resolution profiles of histone modifications.
Using ChIP-seq technology to generate high-resolution profiles of histone modifications.
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DOI：
10.1007/978-1-61779-316-5_20
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发表时间：
2011
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影响因子：
--
通讯作者：
Farnham, Peggy J
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文献类型：
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作者：
O'Geen, Henriette;Echipare, Lorigail;Farnham, Peggy J
DNA和组蛋白的动态修饰通过改变DNA的包装和修饰核小体表面而在转录调控中起关键作用。这些染色质状态，也被称为表观基因组，对于不同的组织、发育阶段和疾病状态是独特的，并且也可以通过环境影响而改变。新技术使表观基因组中编码的信息在全基因组范围内可视化。例如，染色质免疫沉淀（ChIP）试验允许研究人员在体内表征DNA-蛋白质相互作用。ChIP随后与微阵列杂交（ChIP-chip）或通过高通量测序（ChIP-seq）都是鉴定转录因子、组蛋白修饰、DNA甲基化和核小体定位的全基因组谱的强大工具。ChIP-seq技术现在可以仅用一条测序通道以高分辨率询问整个人类基因组，最近已经超过了ChIP芯片技术用于表观基因组分析。重要的是，对于原代细胞和组织的研究，表观遗传谱可以使用少至1 μg的染色质生成。在本章中，我们详细描述了手动或使用IP-Star ChIP机器人进行ChIP检测（重点是表征原代细胞中的组蛋白修饰）所涉及的步骤，然后详细介绍了为Illumina测序准备成功文库的方案。关键的质量控制检查点进行了讨论。尽管这不是本章的重点，但我们也向读者介绍了几种方法，通过这些方法可以分析大量ChIP-seq数据集以提取其中包含的大量信息。
The dynamic modification of DNA and histones plays a key role in transcriptional regulation through altering the packaging of DNA and modifying the nucleosome surface. These chromatin states, also referred to as the epigenome, are distinctive for different tissues, developmental stages, and disease states and can also be altered by environmental influences. New technologies allow the genome-wide visualization of the information encoded in the epigenome. For example, the chromatin immunoprecipitation (ChIP) assay allows investigators to characterize DNA–protein interactions in vivo. ChIP followed by hybridization to microarrays (ChIP-chip) or by high-throughput sequencing (ChIP-seq) are both powerful tools to identify genome-wide profiles of transcription factors, histone modifications, DNA methylation, and nucleosome positioning. ChIP-seq technology, which can now interrogate the entire human genome at high resolution with only one lane of sequencing, has recently surpassed ChIP-chip technology for epigenomic analyses. Importantly, for the study of primary cells and tissues, epigenetic profiles can be generated using as little as 1 μg of chromatin. In this chapter, we describe in detail the steps involved in performing ChIP assays (with a focus on characterizing histone modifications in primary cells) either manually or using the IP-Star ChIP robot, followed by a detailed protocol to prepare successful libraries for Illumina sequencing. Critical quality control checkpoints are discussed. Although not a focus of this chapter, we also point the reader to several methods by which massive ChIP-seq data sets can be analyzed to extract the tremendous information contained within.