siRNA transfection

U2OS cells were plated in 1 ml of DMEM at 4 × 10⁴ per well in 6-well plates. The first round of siRNA transfection was performed right after the plating when the cells were still floating. The transfection mix was made as follows: 200 μl Opti-MEM (Thermo 11058021) were added to a sterile 1.7 ml Eppendorf tube, then 1.2 μl of siRNA (5 μM stock) was added, followed by 2 μl of Lipofectamine RNAi-MAX (Invitrogen 13778-075). The tube was gently mixed and incubated at room temperature for 15 min. This mixture was added dropwise in the well containing the cells. The next day the media was replaced with 1 ml of fresh media and the transfection was repeated one more time. The cells were analyzed 96 h after the first transfection. The final siRNA concentration in the media was 5 nM. The pools of 30 siRNAs targeting ASH2L (ref# 9070), and the siRNA negative control (ref# si-C005-NEG001, a pool of 30 nontargeting siRNA sequences), were purchased from the siTOOLS Biotech company.