GSM8689855: Hep3B Prox1 OE rep 2; Homo sapiens; ATAC-seq
Cells on culture plates were washed twice with PBS and detached by Accutase digestion (4-6 min, RT) followed by ATAC processing. Cell suspensions were placed into an equal volume of MEF medium followed by centrifugation (500g, 5 min, 4°C) before resuspension in ice-cold PBS + 1% BSA. Cells were then strained through a 70 μm filter, centrifuged (500g, 5 min, 4°C), and 50,000 cells resuspended in 50 μL of ice-cold cell lysis buffer containing 0.1% NP-40 and 0.01% digitonin in wash buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, and 1% BSA in ddH2O) for 3-5 minutes. 1 mL of ice-cold wash buffer was then added to stop cell lysis, and nuclei were centrifuged (500g, 10 min, 4°C). 4.5 μL of each of the tagmentation oligos, Tn5_ME and Tn5-R1N, were annealed in oligo annealing buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl, and 10 mM EDTA final concentration in ddH2O) by heating at 95°C (3 min) followed by a ramp down by 1°C to 25°C. The same procedure was performed for Tn5_ME and Tn5-R2N. Tn5 was assembled with annealed oligos by combining 50 μL Tn5 (1 mg/mL stock) with 25 μL annealed Tn5_ME and Tn5-R1N and 25 μL Tn5_ME and Tn5-R2N. Nuclei were resuspended in 40 μL tagmentation buffer (38.8 mM Tris-acetate, 77.6 mM K-acetate, 11.8 Mg-acetate, 18.8% dimethylformamide, and 0.12% NP-40 in ddH2O), to which 5 μL ice-cold PBS + 1% BSA and 5 μL pre-assembled Tn5 was added. Samples were incubated on a Thermomixer (37°C, 30 min, 500 rpm) before being subjected to MinElute (Qiagen) cleanup to extract tagmented DNA. Eluted DNA was pre-amplified with P5 and P7 primers using the NEBNext HF 2x PCR Master Mix (New England Biolabs) in a thermocycler set to 72°C (5 min), 98°C (30 sec), and 5 cycles of 98°C (10 sec), 63°C (30 sec), and 72°C (1 min). A qPCR side reaction was performed with the resulting pre-amplified libraries to determine the necessary additional cycles (5 cycles fewer than the number of cycles corresponding to ⅓ of max fluorescence) for complete amplification. After finishing amplification, 50 μL of each library was subjected to 2-sided size selection by addition of 27.5 μL (0.55x) AMPure XP beads, incubation (5 min), transfer of supernatant to new tubes, addition of 42.5 μL (1.4x) AMPure XP beads to the supernatant, incubation (5 min), three washes with 80% ethanol, and elution of DNA.