Cell Culture and Transfection Protocols

HEK 293T and BHK cells were cultured in DMEM supplemented with 10% fetal calf serum and 50 μg/ml gentamycine (PAA). MEF cells were cultured in DMEM supplemented with 15% fetal calf serum, 0.1 mM β-mercaptoethanol (Invitrogen), 2 mM l-glutamine, 1× MEM non-essential amino acids, 100 U/ml penicillin and 100 g/ml streptomycin (PAA). ESCs including J1 wt, Dnmt1^−/−, E14 wt and Uhrf1^−/− were cultured without feeder cells in gelatinized flasks as described^{33 (link)}. Culture medium was supplemented with 1 000 U/ml recombinant leukemia inhibitory factor (Millipore). The Dnmt1^−/− ESCs used in this study are homozygous for the c allele^{68 (link)}. Mouse E14 wt and Uhrf1^−/− cells have been reported before^{61 (link)}. Mouse ESCs and MEF cells were transfected with FuGENE HD (Roche), Lipofectamine® 2 000 or 3 000 reagent (Invitrogen) according to the manufacturer's instructions. HEK 293T cells and BHK cells were transfected using polyethylenimine as transfection reagent (Sigma) according to the manufacturer's instructions. Cell fixation and microscopy were carried out as described^{35 (link)}.