Control of 5C
Bacterial artificial chromosomes (BACs) covering the Pcdh clusters were also digested with HindIII as a control. The DNAs were then ligated with T4 DNA ligase and purified with phenol-chloroform. 5 ng of BAC library was mixed with 1.5 g of salmon testis DNA. 7 fmol of each 5C primer and 1 l of 105C annealing buffer (NEBuffer 4) were added into the samples to make a total volume of 10 l. Denaturing was performed by incubating samples at 95 C for 5 min followed by annealing at 48 C for 16 h. The DNA was then ligated by Taq DNA ligase (NEB) in the Taq DNA Ligase Reaction Buffer (NEB) at 48 C for 1 h followed by incubation at 65 C for 10 min to stop the reaction. 6 l of ligated products was amplified by PCR using KOD-Plus-Neo (TOYOBO) with specific primers (Table S1) in 25 ul of volume and purified with MinElute PCR Purification Kit (QIAGEN).