2.5 5hmC and 5mC quantification by LC-MS/MS

The 5hmC and 5mC content in cells were quantified by the LC-MS/MS using a stable isotope-labeled internal standard as described previously (Le et al., 2011; Zhang et al., 2016). Briefly, genomic DNA was extracted using DNA extraction kits (Life Technologies, United States) according to the manufacturer’s protocol. Enzymatic digestion of DNA was performed with dsDNA Shearase (Zymo Research, United States). The nucleoside mixture from DNA was oxidized in a buffer containing acetonitrile, formic acid, and MnO₂ and incubated at 40°C for 1 h. The mixture was then purified by a carbon black-SPE column to remove salt, followed by derivatization with dansylhydrazine (DNSH). The labeled products were then reconstituted in water and subjected to UHPLC-MS/MS analysis.

The UHPLC-MS/MS analysis was performed on an Agilent 6460 Triple Quadrupole LC/MS System with a Waters Acquity BEH C18 column. A mobile phase consistent of A (water containing 0.1% formic acid) and B (acetonitrile containing 0.1% formic acid) was used following a gradient elution protocol. The follow rate was set as 0.3 ml/min with 3-µl injection volume. The multiple reaction monitor was used for the mass spectrometer with m/z 242.1→126.1 for 5mC, 258.1→142.1 for 5hmC, and 228.1→112.1 for dC. Collision energy and fragmentor were 33 V and 185 V, respectively.