Lentivirus production, infection, and generation of cell lines

Lentivirus were generated by transfecting HEK293T cells with standard packaging vectors using TransIT-LT1 Transfection Reagent (Mirus Bio, Madison, WI) or Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Briefly, HEK293T were plated in a six-wells plate on day 0 (0.5 × 10⁶ cells per well) and transfected on day 1 with a liposome/DNA mixture containing the following packaging plasmids (0.1 µg of pGag/Pol, 0.1 µg of pREV, 0.1 µg of pTAT, and 0.2 µg of pVSVG) and 1.5 µg of lentiviral vector. On days 3 and 4, the media was replenished with 3 mL of fresh DMEM + glucose media. On days 4 and 5, the viral suspensions were harvested, pooled, pelleted at 1000 g for 5 min, and the supernatant was filtered through 0.45 μm PES filters (Thermo Scientific, cat# 725–2545). The viral suspension was either used directly or kept frozen at −80°C until transduction. For transduction, U2OS and K562 cells were plated in six-well plates (175,000 and 200,000 cells/well, respectively) and infected with 0.5–2 mL of viral suspension supplemented with polybrene at a final concentration of 8 µg/mL. Infected cells were grown for several days before selection with antibiotics or FACS.

K562 dCas9-KRAB cells were previously published (Gilbert et al., 2013) and authenticated by ATCC using STR profiling. U2OS dCas9-KRAB cells were generated by lentiviral transduction of U2OS cells obtained from ATCC (HTB-96) with pMH0006 (Addgene, cat# 135448; Chen et al., 2019) and selected for BFP expression by FACS. CRISPRi knockdown and control cell lines were generated by subsequent lentiviral transduction of dCas9 lines with plasmids containing individual sgRNAs (pOCIAD1sgRNA1 or pOCIAD1sgRNA2) or a non-targeting sgRNA and selected for higher levels of BFP expression by FACS. OCIAD1 knockdown cell lines rescued with wildtype or F102A OCIAD1 were generated by lentiviral transduction with plasmids pUltra-OCIAD1 and pUltra-OCIAD1(F102A), respectively, and selected for GFP expression by FACS. The U2OS OCIAD2 shRNA knockdown cells were generated by lentiviral transduction with plasmids containing individual shRNAs and selected with 15 µg/mL blasticidin for 7 days. The U2OS OCIAD1/2 double knockdown cell line was generated by infecting stable U2OS CRISPRi cells stably expressing sgRNA#2 (above) with the lentivirus vector pLKO1-OCIAD2_shRNA1 and selecting infected cells with 15 µg/mL blasticidin for 7 days. A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15 µg/mL blasticidin for 7 days. Cell lines expressing truncated OCIAD1 constructs were generated by lentiviral infection of CRISPRi cells stably expressing sgRNA#2 (above) with the indicated pFUGW-OCIAD1 and pUltra-OCIAD1-TEV-StrepII lentiviral vectors. U2OS cells stably expressing matrix- or IMS-targeted GFP_1–10 were generated by lentiviral transduction with the plasmids pMTS-GFP1-10 and pIMS-GFP1-10, respectively.