GSM5946870: GPNMB_4C; Homo sapiens; OTHER
The fixed cells were then lysed with a cell lysis buffer, which contains 50 mM Tris-Cl pH7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100 and 1x protease inhibitor cocktail (Roche, Cat#11873580001), for 20-30 minutes on ice. After lysing, the nuclei were harvest and washed with 1x restriction enzyme buffer once. The nuclei pellets were then resuspended in 1x restriction enzyme buffer and treated with 0.3% SDS for 1 hour at 37°C under shaking, followed by another hour with 2.5% Triton X-100. Chromatin digestion was then done by incubating the samples with designated restriction enzyme at proper temperature for overnight while rotating in an airbath. The restriction enzymes used for each locus were listed in "VPinfo.txt". After digestion, heat inactivation at 65°C was applied to inactivate the enzymes and the nuclei were then subjected to ligation with 50μl of T4 DNA ligase (Invitrogen, Cat# 15224-090) in a 7ml ligation solution at 16°C for overnight. Reverse-linking was then performed by treating the samples with proteinase K to get the proximity ligated DNA. The purified DNAs were quantified and subjected to the secondary restriction enzyme digestion, roughly 1 unit of restriction enzyme for 1μg DNA, at the suggested temperature for overnight. After inactivation of the restriction enzymes, the samples were then self-ligated with T4 DNA ligase. The ligated DNAs were recovered with Sodium Acetate and ethanol and quantified with Qubit dsDNA HS assay kit (Thermo Fisher, Cat# Q32851). The 4C-templates were then amplified with designed primers to generate libraries for sequencing. Firstly, the 4C templates were amplified with locus specific primers using 200ng template in each reaction, and we combined 5 reactions for each locus. A 50 μl PCR product aliquots were purified with homemade seramega beads. And one fifth of the purified DNAs were used for the second PCR. The final products were then purified and subjected to sequencing.