Matelot Mélody, Noordermeer Daan
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris Sud, University Paris-Saclay, 1 Avenue de la terrasse, 91198, Gif sur Yvette, France.
Methods Mol Biol. 2016;1480:223-41. doi: 10.1007/978-1-4939-6380-5_20.
3D chromatin organization is essential for many aspects of transcriptional regulation. Circular Chromosome Conformation Capture followed by Illumina sequencing (4C-seq) is among the most powerful techniques to determine 3D chromatin organization. 4C-seq, like other modifications of the original 3C technique, uses the principle of "proximity ligation" to identify and quantify ten thousands of genomic interactions at a kilobase scale in a single experiment for predefined loci in the genome.In this chapter we focus on the experimental steps in the 4C-seq protocol, providing detailed descriptions on the preparation of cells, the construction of the circularized 3C library and the generation of the Illumina high throughput sequencing library. This protocol is particularly suited for the use of mammalian tissue samples, but can be used with minimal changes on circulating cells and cell lines from other sources as well. In the final section of this chapter, we provide a brief overview of data analysis approaches, accompanied by links to publicly available analysis tools.
三维染色质组织对于转录调控的许多方面至关重要。环状染色体构象捕获结合Illumina测序（4C-seq）是确定三维染色质组织的最强大技术之一。与原始3C技术的其他改进方法一样，4C-seq利用“邻近连接”原理，在单个实验中以千碱基规模识别和量化基因组中预定义位点的数万个基因组相互作用。在本章中，我们重点介绍4C-seq实验方案中的实验步骤，详细描述细胞制备、环化3C文库构建以及Illumina高通量测序文库的生成。该方案特别适用于哺乳动物组织样本，但经过最小程度的修改也可用于其他来源的循环细胞和细胞系。在本章的最后部分，我们简要概述了数据分析方法，并提供了公开可用分析工具的链接。
