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0 Q&A 8075 Views Aug 5, 2018
Chromosome conformation capture sequencing (Hi-C) is a powerful method to comprehensively interrogate the three-dimensional positioning of chromatin in the nucleus. The development of Hi-C can be traced back to successive increases in the resolution and throughput of chromosome conformation capture (3C) (Dekker et al., 2002). The basic workflow of 3C consists of (i) fixation of intact chromatin, usually by formaldehyde, (ii) cutting the fixed chromatin with a restriction enzyme, (iii) religation of sticky ends under diluted conditions to favor ligations between cross-linked fragments or those between random fragments and (iv) quantifying the number of ligations events between pairs of genomic loci (de Wit and de Laat, 2012). In the original 3C protocol, ligation frequency was measured by amplification of selected ligation junctions corresponding to a small number of genomic loci (‘one versus one’) through semi-quantitative PCR (Dekker et al., 2002). The chromosome conformation capture-on-chip (4C) and chromosome conformation capture carbon copy (5C) technologies then extended 3C to count ligation events in a ‘one versus many’ or ‘many versus many’ manner, respectively. Hi-C (Lieberman-Aiden et al., 2009) finally combined 3C with next-generation sequencing (Metzker, 2010). Here, before religation sticky ends are filled in with biotin-labeled nucleotide analogs to enrich for fragments with a ligation junction in a later step. The Hi-C libraries are then subjected to high-throughput sequencing and the resultant reads mapped to a reference genome, allowing the determination of contact probabilities in a ‘many versus many’ way with a resolution that is limited only by the distribution of restriction sites and the read depth. The first application of Hi-C was the elucidation of global chromatin folding principles in the human genome (Lieberman-Aiden et al., 2009). Similar efforts have since been carried out in other eukaryotic model species such as yeast (Duan et al., 2010), Drosophila (Sexton et al., 2012) and Arabidopsis (Grob et al., 2014; Wang et al., 2015; Liu et al., 2016). Other uses of Hi-C include the study of chromatin looping at high-resolution (Rao et al., 2014; Liu et al., 2016), of chromatin reorganization along the cell cycle (Naumova et al., 2013) and of differences in chromatin organization in mutant individuals (Feng et al., 2014). The tethered conformation capture protocol (TCC) (Kalhor et al., 2011) described here is a variant of the original Hi-C method (Lieberman-Aiden et al., 2009) and was adapted to Triticeae.
2 Q&A 11577 Views Dec 5, 2017
Chromosome conformation capture (3C) techniques are crucial to understanding tissue-specific regulation of gene expression, but current methods generally require large numbers of cells. This protocol describes two new low-input Capture-C approaches that can generate high-quality 3C interaction profiles from 10,000-20,000 cells, depending on the resolution used for analysis.



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