title: 5C of p0wt_brain
design: The brain of p0 mouse was dispersed by 0.0125%  collagenase (Sigma) treatment in DMEM supplemented with 10% FBS for 20 min at 37 C with rotating at 700 rpm. Cells were then filtered through a 100 um cell strainer (BD) to obtain single-cell suspension.Single cells were crosslinked with 1% formaldehyde for 10 min at room temperature. The crosslinked DNA was lysed and digested with HindIII. The DNAs were then ligated with T4 DNA ligase and purified with phenol-chloroform. 500 ng of the ligated DNA was mixed with 1 g of salmon testis DNA (Sigma). 1.7 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 in 25 ul of volume and purified with MinElute PCR Purification Kit (QIAGEN).