Snake venom phosphodiesterase 1

Genomic DNA extraction was performed with a Genomic DNA Purification Kit (Promega) according to the manufacturer's instructions. The concentration of extracted DNA was determined with NanoDrop 2000 (Thermo Scientific) and quality was evaluated with the ratio of absorbance at 260 nm and 280 nm.
To obtain mononucleosides, 5 μg DNA was digested with 2 U calf intestinal phosphatase, 1 U DNase I and 0.005 U snake venom phosphodiesterase I (New England Biolabs) at 37°C overnight. To remove enzymes used for digestion, DNA solution was filtered with ultra-filtration tubes with molecule weight cutoff at 3 KDa (Pall). The filter solution was analyzed with UHPLC-MS/MS for 5mC, 5hmC and 5fC.

Genomic DNA was extracted from the harvested cells using a Genomic DNA Purification Kit (Solarbio, China) according to the manufacturer's instructions. The concentration and quality of the extracted DNA were evaluated by measuring the absorbance at 260 nm and 280 nm. The extracted DNA was digested with 1 U DNase I, 2 U calf intestinal phosphatase and 0.005 U snake venom phosphodiesterase I (New England Biolabs, Ipswich, MA, USA) at 37 °C for 24 h. Proteins from the DNA digestion system were removed prior to the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, using the microcon centrifugal filter device (Millipore, Bedford, MA, USA) with the 3,000 D cutoff membrane through centrifugation at 10,000 g for 30 min.