Global 5hmC quantification by LC-MS/MS

Genomic DNA from fast muscle was isolated from all 12 individuals (6 D and 6 W) using the Quick-DNA miniprep Plus kit (Zymo Research). The concentration of DNA was determined using the Invitrogen Qubit 3.0 fluorometer (ThermoFisher Scientific, USA). Quality and integrity of the DNA was assessed using the NanoDrop 1000 Spectrophotometer (ThermoFisher Scientific) and the Tapestation 2200 (Agilent Technologies, USA), with absorbance ratios 260/280 > 1.93, 260/230 > 1.89 and minimum DNA integrity number (DIN) of 8.4, respectively. For the global quantification of 5hmC, the internal standard d3-5-hydroxymethyl-2ʹ-deoxycytidine (Toronto Research Chemicals, Canada) was added to the DNA samples. Subsequently, they were hydrolysed completely to deoxynucleosides by 20 U benzonase (Santa Cruz Biotech, USA), 0.2 U nuclease P1, and 0.1 U alkaline phosphatase (Sigma Aldrich) in 10 mM ammonium acetate pH 6.0 and 1 mM magnesium chloride at 40°C for 1 hour. Three volumes of acetonitrile were added to the mix before their centrifugation (16,000 g, 30 min, 4°C). The supernatants were dried and dissolved in 50 µl water for LC-MS/MS analysis of modified and unmodified deoxyribonucleosides. Chromatographic separation was performed using an Agilent 1290 Infinity II UHPLC system with an ZORBAX RRHD Eclipse Plus C18 150 × 2.1 mm ID (1.8 μm) column protected with an ZORBAX RRHD Eclipse Plus C18 5 × 2.1 mm ID (1.8 µm) guard column (Agilent Technologies). The mobile phase consisted of water and methanol (0.1% formic acid was added to both phases). For the initial run of 5-hm(dC), the flow rate was set at 0.15 ml/min with 5% methanol for 30 seconds, which was adjusted to a gradient of 5-15% methanol for the next 4 minutes. The flow rate was increased to 0.25 ml/min in a methanol gradient of 15-90% for 3 minutes, and finally re-equilibration was performed at 5% methanol for 4 minutes. A portion of each sample was diluted for the analysis of unmodified deoxynucleosides. Unmodified deoxynucleosides were chromatographed isocratically with 20% methanol. Mass spectrometric detection was performed using an Agilent 6495 Triple Quadrupole system operating in positive electrospray ionization mode, monitoring the mass transitions 261.1/145.1 (d3-5-hm(dC)), 258.1/142.1 (5-hm(dC)), 252.1/136.1 (dA), 228.1/112.1 (dC), 268.1/152.1 (dG), and 243.1/127.1 (dT).