Dneasy microdna extraction kits

DNA was isolated from the adipocytes and bone marrow using DNEasy microDNA extraction kits (QIAGEN). After isolation, samples were hydrolyzed overnight at 37°C in an enzyme cocktail containing S1 nuclease and phosphatase enzymes. Following hydrolysis, 100 μL of pentafluorobenzyl hydroxylamine hydrochloride (1 mg/mL) and 75 μL of glacial acetic acid were added to each sample followed by incubation at 100°C for 30 minutes. After cooling, 2 mL of acetic anhydride and 100 μL of 1-methylimidazole were added followed by 100°C heat block for 5 minutes, then allowed to cool. Afterwards, 3 mL of HyClone water was added to each sample, vortexed, and let sit for 10 minutes. Then, 2 mL dichloromethane were added, and samples were vortexed vigorously for 15 seconds. After centrifugation at 1500 RPM for five minutes, the bottom dichloromethane layer was transferred into a clean 16 x 100 test tube, evaporated to dryness under nitrogen for 30 minutes, followed by a 30 minute dry in a speed vacuum (Labconco, MO, USA) at room temperature.