5C of mus musculus:BAC
As a control, DNA of six bacterial artificial chromosomes (BACs) covering the Pcdh clusters was also digested, ligated, and purified. The control BAC DNA (5 ng) was mixed with 1.5 g of salmon sperm DNA. These samples were then each mixed with 1.7 fmol of each 5C primer and 1 l of 105C annealing buffer (20 mM Tris-acetate pH7.9, 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM DTT) in a total volume of 10 l and denatured at 95for 5 min. Annealing was performed by incubation at 48 C for 16 hr. The annealed DNA was ligated by adding Taq DNA ligase (NEB) in the 5C ligation buffer (25 mM Tris-HCl pH7.6, 31.25 mM potassium acetate, 12.5 mM magnesium acetate, 1.25 mM NAD, 12.5 mM DTT and 0.125% Triton X-100). The ligation reaction was performed for 1 hr at 48 C followed by incubation for 10 min at 65 C to stop the ligation reaction. The ligated products were amplified by PCR with Illumina primer pairs and purified with PCR purification kit (QIAGEN). 5C libraries were sequenced with the 90 bp pair-end mode by Hi-seq 2500 platform of Illumina. All 5C experiments were performed with biological replicates.