Bolduc Nathalie, Lehman Alisa P, Farmer Andrew
Takara Bio USA, Inc. (formerly Clontech Laboratories, Inc.), Mountain View, California.
Curr Protoc Mol Biol. 2016 Oct 10;116:7.28.1-7.28.26. doi: 10.1002/cpmb.24.
Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) has become the gold standard for mapping of transcription factors and histone modifications throughout the genome. However, for ChIP experiments involving few cells or targeting low-abundance transcription factors, the small amount of DNA recovered makes ligation of adapters very challenging. In this unit, we describe a ChIP-seq workflow that can be applied to small cell numbers, including a robust single-tube and ligation-free method for preparation of sequencing libraries from sub-nanogram amounts of ChIP DNA. An example ChIP protocol is first presented, resulting in selective enrichment of DNA-binding proteins and cross-linked DNA fragments immobilized on beads via an antibody bridge. This is followed by a protocol for fast and easy cross-linking reversal and DNA recovery. Finally, we describe a fast, ligation-free library preparation protocol, featuring DNA SMART technology, resulting in samples ready for Illumina sequencing. © 2016 by John Wiley & Sons, Inc.
染色质免疫沉淀（ChIP）结合高通量测序（ChIP-seq）已成为全基因组范围内转录因子和组蛋白修饰图谱绘制的金标准。然而，对于涉及细胞数量少或靶向低丰度转录因子的ChIP实验，回收的DNA量很少，使得衔接子连接极具挑战性。在本单元中，我们描述了一种可应用于少量细胞的ChIP-seq工作流程，包括一种用于从亚纳克量的ChIP DNA制备测序文库的强大单管无连接方法。首先介绍了一个ChIP实验方案，该方案可选择性富集通过抗体桥固定在磁珠上的DNA结合蛋白和交联DNA片段。接下来是一个快速简便的交联逆转和DNA回收方案。最后，我们描述了一种快速、无连接的文库制备方案，其特点是采用DNA SMART技术，可得到准备好用于Illumina测序的样品。© 2016约翰威立父子公司版权所有
