Lentiviral Knockdown of RGC32 in Lymphoblastoid Cells

Lentiviral particles were generated using the GLTR system (34 (link)) by transfecting HEK-293 FT cells (gift from Dr H. Hochegger) with pLK01 scrambled shRNA (gift from Dr H. Hochegger), pLK01 shRNA RGC32.1, or pLK01 shRNA RGC32.2 and packaging vectors pVSV-G and psPAX 2 (gift from Dr H. Hochegger) using Fugene HD (Promega) in 10 cm dishes. Lentiviruses were harvested by pelleting cells, decanting the culture supernatant and filtering it through a 0.45 μm MillexGP filter (Millipore). The cleared supernatants were aliquoted and 1 ml of lentivirus was added to 5 × 10⁶ GM12878 or IB4 cells. Cells were assayed for the constitutive expression of GFP after 5 days by flow cytometry using a BD FACS Canto or BD Accuri C6 (BD Biosciences). shRNA expression was induced by addition of doxycycline at 0.5 μg/ml to 2 × 10⁵ cells in a 24-well plate. Cells were split after 5 days and then every 2 days. Cells were harvested at different time periods after shRNA induction and the number of GFP positive cells determined by flow cytometry as described above. Sorting of GFP-positive cells was performed using a BDFACSAria (BD Biosciences).