Alkaline phosphatase

Genomic DNA extraction from tissues and cultured cells employed a spin column (high pure PCR template preparation kit, Roche Mannheim, Germany) according to the instruction manual. Cauda epididymidal sperm from of 8–12 week old B6D2F₁ males were washed once in PBS prior to DNA extraction. DNA from gametes, embryos and in experiments using 10 to 1000 ES-D3 cells was extracted on ultra-microspin columns (nucleospin tissue XS, Macherey-Nagel GmbH and Co. KG, Duren, Germany) and the total recovered DNA (4.08 ± 0.66 ng from 1000 cells) subjected to small-scale lc-ms/ms (SMM). Genomic DNA digestion was performed as reported with minor modifications54 (link). Briefly, <1 μg DNA was digested at 37 °C for 3 h with nuclease P1 (4U, Wako Pure Chemical Industries, Ltd., Osaka, Japan) and alkaline phosphatase (3U, Wako) in 100 μl of buffer mixture (3 mM sodium acetate [pH 5.3] containing 1 mM 2-mercaptoethanol, 2 mM ZnSO₄) followed by the addition of 20 μl 50 mM Tris-HCl (pH8.5) and continued incubation for 3 h. Enzymes were methanol-precipitated, and the supernatant containing nucleoside evaporated and stored at −80 °C. Each digest was reconstituted in 100 μl of internal standard solution containing 5 mC-d₃ (0.1 nM), 5 hmC-d₂ (0.1 nM) and ¹⁵N₅-G (1 nM) before being subjected to SMM.