Knockdown of SUV39H1 and SUV39H2 in BFFs by siRNA transfection

The siRNA transfection of BFFs was performed according to the methods reported by Matoba et al. [26]. To this end, the siRNAs against bovine SUV39H1 and SUV39H2 (S1 Table) were diluted in nuclease-free water at 50 μM stock solutions. siRNAs were introduced into the BFFs with Lipofectamine 3000 (#3000–001; Life Technologies). Briefly, 1 × 10⁵ BFFs were seeded into a 24-well plate (day 0). After 24 h, the BFFs were transfected with 5 pM siRNAs Lipofectamine 3000 (day 1). After 24 h, the culture media were refreshed (day 2). On day 3, the BFFs were passaged into 24-well plates at the density of 1 × 10⁵ cells. Transfection was repeated once, as described above (day 4). Next, 48 h after the second transfection (day 6), the transfected BFFs were used for further assessments (flow cytometry, RT-qPCR, or SCNT). In addition, to determine the synergistic effect of siSUV39H1/H2 and TSA in SCNT, 24 h after the second transfection (day 5), the transfected BFFs were treated with TSA for 24 h and then used for SCNT.

In a separate experiment, the relative intensity of H3K9me3 was evaluated in both single and double transfected cells using flow cytometry.