TFAP2C-/- UCLA1 hESCs transduced with a construct that allows doxycycline inducible expression of TFAP2C; cells were cultured in primed conditions and induced for 24 hours with doxycycline
TFAP2C-/- 1d TFAP2C Induc TFAP2C ChIP
Ovation Ultralow Library System V2
Sequenced DNA Library
Cells were fixed with 1% paraformaldehyde (Sigma) and incubated with rotation for 10 minutes at room temperature. The paraformaldehyde was quenched by adding glycine to a final concentration of 0.14M and rotated another 10 minutes at room temperature. The cells were then centrifuged 735g for 5 minutes and then flash frozen with liquid nitrogen and stored at -80°C until ChIP was conducted. To lyse the cells for ChIP, cells were thawed and resuspended with 1mL lysis buffer (10mM TrisHCl pH 8.0, 0.25% Triton X-100, 10mM EDTA, 0.5mM EGTA, 1x Protease inhibitors (Roche) and 1mM PMSF), then rotated 15 minutes. Nuclei were pelleted by centrifugation at 1500g for 5 minutes at 4°C. Nuclei were then resuspended with 1mL 10mM TrisHCl pH 8.0, 200mM NaCl, 10mM EDTA, 0.5mM EGTA, 1x Protease inhibitor, 1mM PMSF and rotated 10 minutes. Nuclei were then pelleted and resuspended in 650μL 10mM TrisHCl pH 8.0, 10mM EDTA, 0.5mM EGTA,1x Protease inhibitor, 1mM PMSF and sonicated in a 12mm x 12mm Sonication Tube (Covaris) in a Covaris S2 (Intensity = 5; Cycles/burst = 200; Duty Cycle = 5%; 8x (30” on/30” off) for four minutes effective sonication). The sonicated lysate is then centrifuged for 10 minutes at 14200g, and the supernatant retained. 10% of the supernatant is saved as “Input”, the rest is used for ChIP. 30μL of Protein A Dynabeads (ThermoFisher) are washed three times with ChIP buffer (16.7mM TrisHCl pH 8.0, 0.01% SDS, 1.1 Triton X-100, 1.2mM EDTA, 167mM NaCl), each wash consisting of addition of 1mL of buffer and collection of the beads on a magnetic rack (Diagenode). The 30uL of beads were then resuspended in 650μL of ChIP buffer and combined with the ChIP sample to pre-Clear the sample. Beads and chromatin were rotated 2hrs at 4°C, and the beads were collected and the supernatant retained. 3μL of anti-TFAP2C antibody (Santa Cruz, sc-8977) was added to the ChIP sample. The samples were then rotated overnight at 4°C. 60μL of Protein A Dynabeads were then added to the ChIP samples and rotated 2 hours at 4°C. The beads were then washed 2x4 minutes with 500μL Wash Buffer A (50mM HEPES pH 7.9, 1% TritonX-100, 0.1% Deoxycholate, 1mM EDTA, 140mM NaCl), 500μL Wash Buffer B (50m HEPES pH 7.9, 0.1% SDS, 1% Triton X100, 0.1% Deoxycholate, 1mM EDTA, 500mM NaCl), and 500μL TE buffer (10mM TrisHCl pH 8.0, 1mM EDTA). Each wash consists of resuspension in 500μL buffer and rotation at 4 minutes, followed by collection of beads and removal of supernatant. DNA was eluted in 100μL elution buffer (50mM TrisHCl pH 8.0, 1mM EDTA, 1% SDS) at 65°C for 10 minutes in a ThermoMixer (Eppendorf) shaking at 1400RPM. The eluent was collected and the beads were subjected to a second round of elution with 150μL elution buffer. The ChIP eluants were pooled, and the input sample was diluted to 250μL with elution buffer. These samples was incubated 65°C overnight to promote decrosslinking. The samples were then allowed to cool to room temperature, 15μg of RNase A (Purelink, ThermoFisher) was added, and the samples were incubated 30 minutes at 37°C to degrade RNA. 100 μg Proteinase K was then added and the samples were incubated 56°C for two hours. DNA was purified using a MinElute PCR Purification kit (Qiagen). DNA was sonicated again to 150bp average fragment size with a Covaris S2, concentrated with Agencourt AMPure XP beads (Beckman Coulter) and libraries were generated using the Ovation Ultralow Library System V2 (Nugen).