CRISPR Knockout of CBX8 and EZH2 in GBM Cells

HEK293T cells were cultured in Dubecco's Modified Essential Media (DMEM), 10% fetal bovine serum (FBS, JR Scientific), 1% glutagro (Corning), 1% penicillin/streptomycin (Corning), 1% sodium pyruvate (Corning). GBM T98G cells were cultured in Eagle's Modified Essential Media, 10% FBS, 1% non-essential amino acids (Corning), 1% glutagro (Corning), 1% penicillin/streptomycin (Corning) and 1% sodium pyruvate (Corning). Hs68 cells were cultured in Dubecco's Modified Essential Media, 10% fetal bovine serum (JR Scientific), 1% penicillin/streptomycin (Corning) and 1% glutagro (Corning). All cells were grown at 37°C and 5% CO₂. For generation of CBX8 and control CRISPR knockout lines, 200 000 T98G cells were plated in six-well 24 h prior to transfection. The respective vector (3.3 μg) was co-transfected with 13 μl of Fugene 6 (Promega). Media was changed 24 h post-transfection. Transfected cells underwent puromycin selection (2 μg/ml) for 3 days, 48 h post-transfection. EZH2 knockout lines were transduced with the MSCV_Cas9_puro vector (a gift from Chris Vakoc, Addgene plasmid #65655) (28 (link)) followed by the guide RNA vector.