ChIP-seq INA-6
ChIP-seq INA-6
ChIP was conducted by utilizing the iDeal ChIP-seq Kit for Histones (Diagenode, #C01010051) according to the manufacturer's instructions. In short, 7 million INA-6 cells treated with vehicle DMSO, UNC1999, 5-azacytidine or combination, were cross-linked for 8 min at room temperature in 1% formaldehyde (Thermo Scientific, #28906). Then, 0.1 M of glycine was added to cease the cross-linking reaction. Shearing of chromatin was done in 10 cycles on the PicoBioruptorTM (Diagenode) (30 sec on/30 sec off). Samples were then incubated with either anti-H3K27me3 (5.68µg) (Diagenode, #C15410196), anti-H3K27ac (2µg) (Active Motif, #39155), anti-H3K4me1 (2µg) (Abcam, #8895) or anti-H3K4me3 (2µg) (Millipore, #07-473) antibodies. The MM cell line INA-6 was cultured in RPMI-1640 AQmediaTM media (Sigma) supplemented with 10% heat-inactivated FBS (Sigma), 1% GlutaMAXTM-I and antibiotics (penicillin 100U/ml and streptomycin 50 mg/mL; Sigma) and Interleukin-6 (IL-6) at 37°C in a humidified 5% CO2 in-air atmosphere incubator. Exponentially growing cells were seeded at 100.000 cells/mL and incubated overnight before addition of reagents. Every 72h, cells were split in equal numbers and re-plated. 7 million INA-6 cells treated with vehicle DMSO, 0.5µM UNC1999, 50nM 5-azacytidine or the combination of 0.5µM UNC1999 and 50nM 5-azacytidine for 9 days. iDeal ChIP-seq Kit for Histones (Diagenode, #C01010051) according to the manufacturer's instructions. SMARTer ThruPLEX DNA-seq library preparation kit WITH  fragmentation (Takara Bio).