Synthetic siRNA oligos targeting hsYY1 and non-targeting control oligos were obtained from Guangzhou Ribo Bio Co. Ltd. The transfection of 293 T cells was performed with the indicated siRNA using Lipofectamine® RNAiMAX according to the manual (cat.: 11668-019, Thermo Fisher Scientific). Cells were harvested at 48 h post-transfection. The hsYY1 knockdown efficiency was analyzed by western blotting (using anti-YY1 (H414; sc-1703) or anti-YY1 (C20; sc-281) antibodies, Santa Cruz). Signals were normalized to GAPDH.
For 293TshYY1 cell construction, three pairs of hsYY1 shRNA oligomers were synthesized and annealed according to the manual’s instructions (Thermo Fisher Scientific). The lentiviral pLKO.1-shYY1 plasmids were first constructed. Viral particles were then prepared by transfecting psPAX2 and pMD2.G with pLKO.1-shYY1 plasmids into 293T cells. A more detailed description of the lentiviral package can be obtained from the instructions for Addgene Plasmid 10878. Finally, the constructed 293TshYY1 cells were screened out via puromycin resistance and maintained as usual with puromycin (2 µg mL−1) addition. The hsYY1 knockdown efficiency was confirmed by western blotting. Three pairs of hsYY1 shRNA oligomers are listed in Supplementary Table1 .
For 293TshYY1 cell construction, three pairs of hsYY1 shRNA oligomers were synthesized and annealed according to the manual’s instructions (Thermo Fisher Scientific). The lentiviral pLKO.1-shYY1 plasmids were first constructed. Viral particles were then prepared by transfecting psPAX2 and pMD2.G with pLKO.1-shYY1 plasmids into 293T cells. A more detailed description of the lentiviral package can be obtained from the instructions for Addgene Plasmid 10878. Finally, the constructed 293TshYY1 cells were screened out via puromycin resistance and maintained as usual with puromycin (2 µg mL−1) addition. The hsYY1 knockdown efficiency was confirmed by western blotting. Three pairs of hsYY1 shRNA oligomers are listed in Supplementary Table