GSM3611807: NB4_ChIP_seq_PML_RARA; Homo sapiens; ChIP-Seq
Cells were washed once with PBS buffer and 3 times with lysis buffer (10 mM Tris-HCl, PH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40, 1 mM PMSF). 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 5 μg antibodies against PML/RARα fusion protein. 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.
GEO Accession
GSM3611807