Multiplexed analysis of chromosome conformation at vastly improved sensitivity

Current methods for analysing chromosome conformation in mammalian cells are either insensitive and low resolution or low throughput. Since the methods are expensive and relatively difficult to perform and analyse they are not widely used outside of specialised laboratories. Here we have re-designed the Capture-C method producing a new approach, called next generation (NG) Capture-C, which solves most of the current setbacks in analysing chromosome conformation. NG Capture-C produces unprecedented levels of sensitivity and reproducibility, which can be used to analyse any number of genetic loci and/or many samples in a single experiment. NG Capture is straightforward to perform, requiring only standard reagents and access to basic next generation sequencing platforms. The complete and detailed protocol presented here, with new publically available tools for library design and data analysis, will allow most laboratories to analyse chromatin conformation at levels of sensitivity and throughput that were previously impossible. Overall design: NG Capture-C combines 3C library preparation with oligonucleotide capture for the desired viewpoint restriction fragments, in this case the promoters of genes were exclusively used. The capture step has been optimised to markedly increase the sensitivity of the method (by introducing a double capture step), providing unprecedented levels of sensitivity and reliability for high resolution analysis of the chromatin landscape.