The different steps of the original genomic 3C experiment in yeast and their associated biases . A) Experimental steps. 1: Yeast cells are fixed with formaldehyde. 2: the genome is digested using a 6 cutter restriction enzyme (RE1; red double-headed arrows). 3: extraction of protein/DNA complexes and ligation in diluted conditions that favor DNA-end interactions and religation within the same complex. During this process, some RF will simply circularize (i), while others will religate in their original orientation (ii). Religation products are also expected between non-collinear restriction fragments (iii), whereas collinear RF separated by one, or more, RF will also interact together (iv). 4: de-crosslinking and DNA purification. 5: digestion of DNA products using a frequent 4 cutter restriction enzyme (RE2; black double-headed arrows). 6: DNA is ligated in diluted conditions, favoring intra-molecular circularization of single DNA molecules. Remaining linear fragments are degraded. 7: DNA circles containing a RE1 site are re-opened using RE1. 8: short DNA sequences, containing EcoP15I recognition site and a biotinylated nucleotide are added at both ends of the linear fragments. 9: circularization of linear fragments. 10: EcoP15I digestion of the DNA segments 25 bp apart from the enzyme recognition site. 11: pull-down of the DNA fragments containing biotinylated nucleotides. 12: amplification of the DNA fragment isolated and sequencing. B) Pie-chart representation of the different types of events obtained at step 3: religations, long range intra, long range inter, loops (from 50 millions pair-end sequences analyzed from the HindIII-MspI condition A and B experiments). C) Quantification of the fragment length bias. D) Quantification of the GC bias. E) Quantification of the circularization length bias.
Cournac et al. BMC Genomics 2012 13:436 doi:10.1186/1471-2164-13-436