The lentiviral vector (GV248) carrying shRNA targeting human SMC1A was obtained from GeneChem (shSMC1A, 5'-CGGGACTGTATTCAGTATA-3'; shCont, 5'-TTCTCCGAACGTGTCACGT-3'). Lentivirus was produced in HEK-293T cells using the shRNA-expression GV248 vector with packaging plasmids pHelper1.0 and pHelper2.0, which were co-transfected into HEK-293T cells by lipofectamine 2000 (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. The mediums with lentiviral particles were separately harvested at 24 h, 48 h and 72 h after transfection. To deposit lentiviral particles, the medium supplemented with 5 × PEG-itTM Solution (System Biosciences, USA) was incubated at 4°C for 24 h. The lentiviral particles were then collected by centrifugation at 1500 × g for 30 min and dissolved in Phosphate Buffer Saline (PBS).
HepG2 and Bel7402 cells were seeded into 6-well plates at the concentration of 5 × 104 cell/well. Lentiviral particles with shSMC1A or Negative Control (NC) were added into HepG2 cells after 12 h of incubation. Then, after 24 h of infection, the medium with lentivirus particles was replaced with refresh growth medium. Stable cell lines were selected out in 5 μg/mL puromycin for 4 days.
HepG2 and Bel7402 cells were seeded into 6-well plates at the concentration of 5 × 104 cell/well. Lentiviral particles with shSMC1A or Negative Control (NC) were added into HepG2 cells after 12 h of incubation. Then, after 24 h of infection, the medium with lentivirus particles was replaced with refresh growth medium. Stable cell lines were selected out in 5 μg/mL puromycin for 4 days.