Lentiviral vector construction and transfection

Lentivirus vector for mouse CENPF small hairpin RNA (shRNA) encoding a green fluorescent protein (GFP) sequence was constructed by GENECHEM (Shanghai, China). The target shRNA sequence is 5ʹ-ACTGACCAAACTTGTGATAAA-3ʹ (mouse CENPF gene GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"XM_006497119.4","term_id":"1907066872","term_text":"XM_006497119.4"}}XM_006497119.4). The lentivirus vector was confirmed by sequencing. Negative control shRNA was provided by GENECHEM. Lentivirus-encoded shRNA against CENPF and control were prepared and titered to 2 × 10⁸ (TU/mL) as the manufacturer’s protocol. H22 Cells (1 × 10⁴ cells/well) were seeded in six-well plates overnight before transfection. The virus (0.1 mL) was mixed with 0.1 mL complete medium containing polybrene (8 mg/mL) and added to cells and incubated for 1 h at 37°C. Then the cells were incubated in fresh complete medium containing polybrene for 24 h, followed by incubation for 48 h in complete RPMI-1640 medium. The cells were then harvested and expanded for subsequent studies.