Transient transfections to HEK 293 T cells were performed using polyethyleneimine (PEI) (Polysciences, Warrington, PA, USA) in the OPTI-MEM medium (Life Technologies, Carlsbad, CA, USA) with a ratio of 1:4 to 1:6 of DNA: PEI.
Viral particles were produced by HEK 293 T cells in a 10 cm dish transfected with 4 μg pMD2.G and 6 μg psPAX2 packaging plasmids (Addgene, Watertown, MA, USA), together with 8 μg lentiviral expressing vectors encoding target genes, including pITA -CBX3-N/C-flag, pLKO.1 or hU6-MSC-Ubiquitin-eGFP vector encoding shRNAs targeting interested genes as listed in the Supplementary Information. Supernatant carrying the viral particles was harvested 35 h and 60 h after transfection and concentrated to 100× volume by Poly (ethylene glycol) 8000 (Sigma-Aldrich, St. Louis, MS, USA).
For viral infection, 1 × 106 myeloma cells were seeded in 1 ml new complete media for 6 h and then added 50 μl viral concentration and 8 μg/mL polybrene, and cells were spin at 800 × g for 45 min at 20 °C. In total, 12 h after spinfection, the medium was changed and cells were cultured for another 48 h until further management.
Viral particles were produced by HEK 293 T cells in a 10 cm dish transfected with 4 μg pMD2.G and 6 μg psPAX2 packaging plasmids (Addgene, Watertown, MA, USA), together with 8 μg lentiviral expressing vectors encoding target genes, including pITA -CBX3-N/C-flag, pLKO.1 or hU6-MSC-Ubiquitin-eGFP vector encoding shRNAs targeting interested genes as listed in the Supplementary Information. Supernatant carrying the viral particles was harvested 35 h and 60 h after transfection and concentrated to 100× volume by Poly (ethylene glycol) 8000 (Sigma-Aldrich, St. Louis, MS, USA).
For viral infection, 1 × 106 myeloma cells were seeded in 1 ml new complete media for 6 h and then added 50 μl viral concentration and 8 μg/mL polybrene, and cells were spin at 800 × g for 45 min at 20 °C. In total, 12 h after spinfection, the medium was changed and cells were cultured for another 48 h until further management.
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