GSM8392935: KPC ATAC; Mus musculus; ATAC-seq
PDAC tumors were harvested from two female KPC and two female AKPC mice and snap-frozen. 20-30 mg tissue samples were resuspended in 1ml of wash buffer (0.25 M sucrose, 25 mM KCI, 5 mM MgCl2, 10 mM Tris-HCI (pH 7.5), 1 mM DTT (Sigma, D9779), 1X protease inhibitor (Fischer, PIA32965), 1 U µl−1recombinant RNAsin (Promega, PAN2515), 0.1% IGEPAL (Sigma, I8896) in molecular biology water) and then homogenized using a Dounce homogenizer with 2 pestles. First, a loose pestle was used for 5-10 strokes to break the tissue into small pieces and then a tight pestle was used for 15-25 strokes to fully homogenize the sample into a uniform solution. Homogenized tissue was filtered through a 30 μm filter (CellTrics, Sysmex) and then centrifuged with a swinging-bucket centrifuge (100 rcf, 10 min, 4˚C, Eppendorf, 5920 R). Nuclei were resuspended in 1 ml of wash buffer (0.25 M sucrose, 25 mM KCI, 5 mM MgCl2, 10 mM Tris-HCI (pH 7.5), 1 mM DTT (Sigma, D9779), 1X protease inhibitor (Fischer, PIA32965), 1 U µl−1recombinant RNAsin (Promega, PAN2515) in molecular biology water) and centrifuged again (100 rcf, 10 min, 4˚C, Eppendorf, 5920 R). Next, nuclei were resuspended in 400 µl of sorting buffer (1X protease inhibitor (Fischer, PIA32965), 1 U µl−1recombinant RNAsin (Promega, PAN2515), 1% fatty acid free BSA (Proliant, 7500804), 2uM 7-AAD (Invitrogen, A1310) in PBS (Corning, 21-040-CV)) and incubated on ice for 10 minutes. 120,000 nuclei were sorted into a Lobind tube with 90 µl collection buffer (5 U µl−1recombinant RNAsin (Promega, PAN2515), 5% fatty acid free BSA (Prolian, 7500804) in PBS (Corning, 21-040-CV) using an SH800 cell sorter (Sony). The resulting nuclei suspension was centrifuged again (500 rcf, 5 min, 4˚C, Eppendorf, 5920 R) and resuspended in 100 µl of collection buffer (1% fatty acid free BSA (Proliant, 7500804), 1X protease inhibitor (Fischer, PIA32965), 10 mM Tris-HCI (pH 7.4), 0.1% Tween-20 (Sigma, P7949-100ML), 0.1% IGEPAL (Sigma, I8896), 0.01% digitonin (Promega, G9441), 1mM DTT (Sigma, D9779), 1 U 