Abstract
Chromosome conformation capture (3C) assays are used to map chromatin interactions genome-wide. Chromatin interaction maps provide insights into the spatial organization of chromosomes and the mechanisms by which they fold. Hi-C and Micro-C are widely used 3C protocols that differ in key experimental parameters including cross-linking chemistry and chromatin fragmentation strategy. To understand how the choice of experimental protocol determines the ability to detect and quantify aspects of chromosome folding we have performed a systematic evaluation of 3C experimental parameters. We identified optimal protocol variants for either loop or compartment detection, optimizing fragment size and cross-linking chemistry. We used this knowledge to develop a greatly improved Hi-C protocol (Hi-C 3.0) that can detect both loops and compartments relatively effectively. In addition to providing benchmarked protocols, this work produced ultra-deep chromatin interaction maps using Micro-C, conventional Hi-C and Hi-C 3.0 for key cell lines used by the 4D Nucleome project.
Highlights
Chromosome conformation capture (3C)-based assays[1] have become widely used to generate genome-wide chromatin interaction maps[2]
We selected three cross-linkers widely used for chromatin: 1% formaldehyde (FA), conventional for most 3C-based protocols; 1% FA followed by incubation with 3 mM disuccinimidyl glutarate; and 1% FA followed by incubation with 3 mM ethylene glycol bis(succinimidylsuccinate) (Fig. 1a)
We analyzed four different cell types: pluripotent H1 human embryonic stem cells (H1-hESCs), differentiated endoderm (DE) cells derived from H1-hESCs, fully
Summary
Chromosome conformation capture (3C)-based assays[1] have become widely used to generate genome-wide chromatin interaction maps[2]. Analysis of chromatin interaction maps has led to detection of several features of the folded genome. Such features include precise looping interactions (at the 0.1–1 Mb scale) between pairs of specific sites that appear as local dots in interaction maps. Micro-C, which uses MNase instead of restriction enzymes as well as a different cross-linking protocol, was shown to allow generation of nucleosome-level interaction maps[7,8,9]. It is critical to ascertain how key parameters of these 3C-based methods, including cross-linking and chromatin fragmentation, quantitatively influence the detection of chromatin interaction frequencies and the detection of different chromosome folding features that range from local looping between small intra-chromosomal (cis) elements to global compartmentalization of megabase-sized domains. We systematically assessed how different cross-linking and fragmentation methods yield quantitatively different chromatin interaction maps
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