Dnase free rnase a

Primary cells were isolated incubating mouse embryonic skins on dispase (0.5 ml drops) (CELLnTEC) for 1 h at room temperature. After removal of the dermis, the epidermis was incubated under agitation in cold 0.25% trypsin (2 ml) for 5 min at 4°C and filtered through 100-micron cell strainers. Trypsin action was stopped by adding 3 ml DMEM plus 10% FCS. The cells were centrifuged for 5 min at 259 g at 4°C. The cells were resuspended in a mixture of ice-cold PBS and 70% ethanol and placed on ice for 2 h. The cells were then centrifuged for 5 min at 180 g and the ethanol solution was removed. After an additional wash in PBS, the cell pellet was suspended in a 1-ml solution containing 0.1% Triton X-100 (EMD Millipore) in PBS, 2 mg DNase-free RNase A (QIAGEN) and 200 ml of 1 mg/ml propidium iodide (Sigma-Aldrich) and incubated at room temperature for 30 min. Acquisition was then performed on these samples using a FACSCalibur analyzer (BD) with a minimum of 1 × 10⁴ cells analyzed with CellQuest Pro software (BD).

Formalin-fixed, paraffin-embedded (FFPE) tissues proven to contain tumor sections of the 285 patients were obtained from the pathology department’s archive. For each case, 3–6 sections of 10 μm thickness were taken from the block for the extraction of DNA using the QIAamp DNA FFPE tissue kit (Qiagen, Dusseldorf, Germany), using the manufacturer’s recommended instructions. Briefly, the FFPE sections were deparaffinized using xylene followed by ethanol to extract residual xylene. The specimens are covered with ATL lysis buffer with 20 μL proteinase K (20 mg/mL, Roche, Mannheim, Germany) and incubated at 56 °C and 90 °C for 1 h each. Then, 2 μL 100 mg/mL DNase-free RNase A (Qiagen) was added, mixed and incubated at room temperature for 2 min. After the lysis and heating, followed by binding and washing steps, DNA was eluted in 50 μL of ATE buffer and quantified using a NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).