Figure 1. Overview of the method.

a. Experimental workflow. High-resolution 3C libraries generation:, crosslinking live cells (1); digestion of chromatin, optimized for four cutter restriction enzymes (eg Dpn II) (2); ligation (3); de-crosslinking and DNA extraction (4). This 3C library is sonicated to produce random ~200 bp fragments (5) followed by; sequencing adaptor ligation and indexing (6); pooling of indexed samples (7) hybridization with biotinylated oligonucleotides to the pool of indexed samples (8); pull down using streptavidin beads (9) and PCR from beads using adapter P5&7 sequences (10). Steps 8-10 are repeated, resulting in enrichments up to 3,000,000-fold over the uncaptured 3C library, and sequenced (11).

b. Analysis. 1. Raw data (FASTQ). 2. Reconstruction of paired reads into original fragments. 3. in silico digestion into component restriction fragments to allow for mapping. 4. Reads not containing a restriction site or a captured viewpoint are discarded as background. 5. Reads that are not unique are collapsed into a single representative read. 6. Interactions are only reported if a read pair maps within a captured fragment and maps outside all of the capture fragments and proximity exclusion regions in the experiment (usually 1 kb on either side of the captured viewpoint fragments). This is done to prevent undigested material being reported as interacting and to prevent the reporting of fragments captured by two different oligonucleotides. The data are then filtered to remove regions with problematic mappability due to copy number differences³³ and mis-mapped reads from the proximity exclusion region.