Approximately 3x10^6 Dicer –/– cells were used per transfection and the cells were plated immediately after nucleofection. Transfections of siRNAs were performed using 300 pmol of siRNA against RL mRNA (siRL) (Eurogentec), 50 pmol of siGENOME smartPOOL siRNAs against Rbl2 (Dharmacon), 50 pmol of each of the mmu-mir-290, mmu-mir 291a-3p, mmu-mir-292-3p, mmu-mir-293, mmu-mir-294 and mmu-mir-295 miRNA mimics (Dharmacon), or 300 pmol of mmu-mir-291a-3p, together with 2 mg of pCX-EGFP50, which served as control for transfection efficiency. For rescue of de novo DNA methylation by a mixture of pCag-EGFP-Dnmt3a2, pCag-EGFP-Dnmt3b and pCag-EGFP-Dnmt3L plasmids, the Dicer –/– cells were co-transfected with 7 mg of each of these plasmids. The EGFP-expressing cells were collected using a MoFlow cell sorter after 3 d of culture in the presence of 100 nM RA and the absence of LIF.