Transduction of KDM5B-EGFP-reporter construct

The KDM5B-promoter-EGFP-reporter construct was stably integrated into early passage (p4-p13) naive BN46, BN118, E049, E056, as well as TMZ-exposed ^TMZ→eRBN46, ^cRBN118 patient cell samples via lentiviral infection. Lentivirus was produced in HEK293T cells using the TransIT-TKO® transfection reagent Mirus (Mirus Bio; Madison, Wisconsin, USA), Opti-MEM™ Reduced Serum Medium (Thermo Fisher Scientific; Waltham, MA, USA) and the following plasmids: 2.5μg Lenti (pLU-JARID1Bprom-EGFP-BLAST; from⁹), 2.5μg pMDLg/pRRE (Addgene, Watertown, MA, USA #12251; RRID: Addgene_12251), 2.5μg pRSV-Rev (Addgene #12253; RRID: Addgene_12253); 2.5μg pMD2.G (Addgene #12259; RRID: Addgene_12259). For stable transduction, 10⁶ cells of the indicated patient cell samples were provided with virus for 48 hours and selection of cells was achieved by blasticidin (Invivogen; San Diego, California, USA) and confirmed using flow cytometry for EGFP. The transduced cells were expanded under controlled culture conditions for 2-4 passages, frozen as stocks, thawed and passaged once more for consecutive experimentation.