Acroprep advance 96 filter plate 0.2 μm supor

To measure the cytosine, 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and 8-oxo-7,8-dihydroguanine (8-oxo-G) content of DNA samples, three technical replicates were run for each sample. More specifically, 0.5 to 2 µg DNA in 25 µL H₂O were digested as follows: an aqueous solution (7.5 µL) of 480 μM ZnSO₄, containing 42 units Nuclease S1, 5 units antarctic phosphatase, and specific amounts of labeled internal standards were added and the mixture was incubated at 37 °C for 3 h in a Thermomixer comfort (Eppendorf). After addition of 7.5 μL of a 520 µM [Na]₂-EDTA solution containing 0.2 units snake venom phosphodiesterase I, the sample was incubated for another 3 h at 37 °C. The total volume was 40 µL. The sample was then kept at -20 °C until the day of analysis. Samples were then filtered by using an AcroPrep Advance 96 filter plate 0.2 μm Supor (Pall Life Sciences) and then analyzed by LC-ESI-MS/MS, which are performed using an Agilent 1290 UHPLC system and an Agilent 6490 triple quadrupole mass spectrometer coupled with the stable isotope dilution technique. DNA samples were digested to give a nucleoside mixture and spiked with specific amounts of the corresponding isotopically labeled standards before LC-MS/MS analysis. The nucleosides were analyzed in the positive ion selected reaction monitoring mode (SRM). In the positive ion mode, [M+H]⁺ species were measured.