In vitro methylation of pOct4-GFP plasmid DNA was performed using M.SssI methyltransferase (New England Biolabs) according to the manufacturer's instructions. The methylation status of the plasmid was tested by HpaII and MspI (Fermentas) digestion.
For the in vitro oxidation, GFP-TET1CD or GFP-TET1CDmut (H1652Y, D1654A) was purified from mammalian cells. In detail, HEK293T cells were transfected with an expression construct for GFP-TET1CD/TET1CDmut and immunoprecipitation was carried out using GBP-Ni-NTA beads. Proteins were eluted using imidazole. The in vitro methylated plasmid was diluted in TET reaction buffer (50 mM HEPES pH 8.0, 75 μM Fe(II), 2 mM Sodium-Ascorbate, 1 mM Di-Sodium-Ketoglutarate (39 (link))) and added to the purified GFP-TET1CD.
For the in vitro oxidation, GFP-TET1CD or GFP-TET1CDmut (H1652Y, D1654A) was purified from mammalian cells. In detail, HEK293T cells were transfected with an expression construct for GFP-TET1CD/TET1CDmut and immunoprecipitation was carried out using GBP-Ni-NTA beads. Proteins were eluted using imidazole. The in vitro methylated plasmid was diluted in TET reaction buffer (50 mM HEPES pH 8.0, 75 μM Fe(II), 2 mM Sodium-Ascorbate, 1 mM Di-Sodium-Ketoglutarate (39 (link))) and added to the purified GFP-TET1CD.
