GSM8139577: NB4_overexpression_HA-PML/RARα_IP_HA; Homo sapiens; ChIP-Seq
Cells were fixed with 1% formaldehyde for 10 minutes. After quenching and cell lysis, the pellets were then re-suspended in ChIP buffer (Active motif) containing 1 mM PMSF and proteinase inhibitor cocktail (Sigma). Chromatin shearing was performed using a Biorupter Pico ultrasonicator device (Diagenode) with 30s ON then 30s OFF for 30 cycles. Solubilized chromatin was used for immunoprecipitation by incubating overnight at 4 °C with chip antibodies. The immunoprecipitation reaction was transferred to the Protein G agarose Column (53039, Active Motif) and incubated for 2 hours at 4 °C, then each column was washed 4 times with 900 μL Wash Buffer AM1 (Active motif). The ChIPed sample was eluted using 100 μL 37 °C Elution Buffer AM4 (Active Motif). Cross-linking was reversed by adding 5 μL Proteinase K and the sample was incubated overnight at 65 °C. Input and ChIPed DNA were purified using a DNA purification column (Active Motif). DNA libraries were prepared with the MicroPlex Library Preparation Kit (Diagenode) according to the manufacturer's instructions. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250-450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit.