In Situ Hi-C for Plants: An Improved Method to Detect Long-Range Chromatin Interactions
Recently, long noncoding RNAs (lncRNAs) are shown to be implicating nuclear domain organization and gene regulation by mediating long-range chromatin interactions. Chromosome conformation capture (3C) is a method used to study such long-range interaction between two different loci in the 3D nuclear space. Through successive improvement in resolution and throughput, 3C, chromosome conformation capture on chip (4C), and chromosome conformation capture carbon copy (5C) to Hi-C methods were developed to study interactions between loci from one versus one scale to an unprecedented genome-wide resolution. In situ Hi-C is a variant of Hi-C in which proximity ligation is performed at the intact nuclei to improve the signal-to-noise ratio and throughput of the experiment to provide useful genome-wide contact frequency matrix/maps. The contact frequency maps obtained could be used for physical ordering of scaffolds in complex genome assembly projects, in deducing the nuclear domain organization in high resolution and in identifying specific long-range interactions between genomic regions of interest. In this chapter, we describe in detail a protocol for in situ Hi-C used on crops like barley, wheat, rye, oat, and evening primrose.
Key wordsIn situ Hi-C Chromatin interaction Genome assembly Scaffolding Long noncoding RNA Nuclear domain organization Triticeae
This work was financially supported by core funding of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, and project funding (“SHAPE”) from the German Federal Ministry of Education and Research (BMBF, Grant no. 031B0190A) to Dr. Nils Stein and Dr. Martin Mascher. We thank Dr. Erez Lieberman-Aiden and Dr. Olga Dudchenko for helpful discussions on the in situ Hi-C method. We thank Ines Walde and Manuela Knauft for excellent technical assistance during protocol development and TCC/Hi-C library construction.
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