ATAC-seq to detect open chromatin sites in a selection of ovarian cancer cell lines
ATAC-seq to detect open chromatin sites in a selection of ovarian cancer cell lines
A total of 50,000 viable cells were pelleted at 1,500 rcf and 4 °C for 10 min.  All cells were maintained in a humidified atmosphere at 37°C with 5% CO2. COV-434 cells were cultured in DMEM, 2 mM Glutamine and 10% FBS. ES-2 cells were cultured in McCoy's 5a and 10% FBS. Kuramochi and SKOV-3 cells were cultured in RPMI 1640 and 10% FBS. OAW28 and OAW42 cells were cultured in DMEM, 2 mM glutamine, 1 mM NaP, 5 mg bovine insulin and 10% FBS. OVISE cells were cultured in DMEM and 10% FBS. TOV-112D cells were cultured in 1:1 MCDB 105 medium plus Medium 199 and 15% FBS. Thereafter, the cell pellet was resuspended in 50 µL pre-chilled ATAC-Resuspension buffer (RSB) containing 0.1 % NP-40, 0.1 % TWEEN-20, and 0.01 % digitonin and incubated on ice for 3 min. The lysis was washed out by adding 1 mL cold ATAC-RSB containing only 0.1 % TWEEN-20. Nuclei were pelleted by centrifugation at 500 rcf and 4 °C for 10 min, and the supernatant was completely removed. Illumina Tagment DNA TDE1 (Tagment DNA Enzyme 1) Enzyme and Buffer Kit was used for tagmentation. In brief, the nuclei were resuspended in 47.5 µL transposition mixture (25 µL 2× TD buffer, 16.5 µL PBS, 1 % digitonin, 0.5 µL 10 % TWEEN-20, and 5 µL water) and subsequently 2.5 µL TDE1 was added. The mixture was incubated at 37 °C for 30 min on a thermomixer at 1,000 rpm. Thereafter, the cell pellet was resuspended in 50 µL pre-chilled ATAC-Resuspension buffer (RSB) containing 0.1 % NP-40, 0.1 % TWEEN-20, and 0.01 % digitonin and incubated on ice for 3 min. The lysis was washed out by adding 1 mL cold ATAC-RSB containing only 0.1 % TWEEN-20. Nuclei were pelleted by centrifugation at 500 rcf and 4 °C for 10 min, and the supernatant was completely removed. Illumina Tagment DNA TDE1 (Tagment DNA Enzyme 1) Enzyme and Buffer Kit was used for tagmentation. In brief, the nuclei were resuspended in 47.5 µL transposition mixture (25 µL 2× TD buffer, 16.5 µL PBS, 1 % digitonin, 0.5 µL 10 % TWEEN-20, and 5 µL water) and subsequently 2.5 µL TDE1 was added. The mixture was incubated at 37 °C for 30 min on a thermomixer at 1,000 rpm. The transposed library was PCR amplified using Nextera primers. DNA was purified by adding 70 µL of diluted magnetic AMPureXP beads (10 µL beads and 60 µL bead buffer) and incubating for 10 min at RT. The supernatant was clarified by placing the samples on a magnetic rack and discarded after 10 min of incubation. The beads were washed several times with 75 µL of 80 % ethanol and air-dried for approximately 5−7 min. DNA was eluted by incubating the beads with 12 µL elution buffer (EB) for 2−3 min. To collect the ATAC DNA-containing supernatant, beads and supernatant were separated on a magnetic rack and the supernatant was transferred to a fresh DNA LoBind tube.