Determination of High-Resolution 3D Chromatin Organization Using Circular Chromosome Conformation Capture (4C-seq)

Abstract

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.

Keywords: 3D chromatin organization; 4C-seq; Chromatin compartmentalization; Chromatin loops; Circular Chromosome Conformation Capture; DNA interactions; High-throughput sequencing; Nuclear organization.