title: GSM6541915: D3; Mus musculus; ATAC-seq
library_construction_protocol: For ATAC-seq, 50000 cells were harvested. In the case of OC (D3), 50000 cells were reseeded to a culture plate in Day2 (D2), then the whole plate was used. The cells were washed with PBS and collected in ep tubes. Then, on the ice, the cells were gently suspended and incubated in the ep tubes of 50 μl lysis buffer consisted with 0.1% NP-40, 0.1% Tween-20, and 0.01% Digitonin, and the resuspension buffer containing 10mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, and DW. After the 3 minutes incubation, 1 ml of wash buffer containing 990 μl of the resuspension buffer and 10 μl of 10% Tween-20 was added into the ep tube. Next, the cells were gently washed by inverting the tubes. After this wash step, the cells were centrifugated at 500 ×g for 10 minutes at 4℃. The supernatant was removed, and pelleted nuclei in each tube were gently suspended in transposition solution mix containing 25 μl 2× TD buffer (Illumina), 16.5 μl PBS, 0.1% Tween-20, 0.01 Digitonin, 2.5 μl Tn5 Transposase (Illumina), and 5 μl DW. Then the samples were incubated at 37℃ for 30 minutes on thermomixer at 1000 rpm. Transposed DNA purification was performed using DNA Clean & Concentrator (D4013, Zymo Research). To generate library, barcoded primers  contained in a previous study (Nature Methods volume 10, pages1213–1218 (2013)) were used with NEBNext High-Fidelity 2× PCR Master Mix. The purified DNA was amplified with following conditions: 72℃ 5 minutes, 98℃ 30 seconds, after this the condition next was repeated 5 times, 98℃ 10 seconds 63℃ 30 seconds, 72℃ 60 seconds. To get saturated cycle, qPCR was performed using 100× SYBR Green 1 and 0.5 μl of primers with 5 μl of each PCR product. After getting the maximum R, PCR was conducted with 45 μl PCR product until it reaches 1/3 of the maximum R. The libraries were purified using DNA Clean & Concentrator.