Powerplex 16

A panel of breast cancer cell lines were selected to represent the different molecular classes of breast cancer cells [24 (link)] including: MCF-7, T47D, MDA-MB-453, MDA-MB-468, MDA-MB-231 and BT474, as well as HFFF2 human foreskin fibroblasts, all obtained originally from American Type Culture Collection (Rockville, Maryland, US) and maintained under recommended culture conditions (see S1 Table). To avoid genetic drift early passage cell stocks were used for no more than 3 months, after which they were replaced with frozen stocks from the same original early passage stocks. Quarterly mycoplasma tests (MycoAlert, Lonza, Switzerland) were consistently negative and annual STR testing (Powerplex16, Promega, Madison, Wisconsin, US) confirmed cell identity. Following ethical approval (Leeds East Research Ethics Committee 09/H1036/108) and written consent from donors, primary human mammary fibroblasts were generated in house from normal breast tissue obtained from a reduction mammoplasty [25 (link)]. One of these, LS11-083, was hTERT-immortalised [26 (link)] and ds-Red transduced, to increase longevity and enable tracking in culture, as previously described [16 (link)]. Where measured, cell growth was estimated using a haemocytometer.

The RME cell line RD (ATCC, Manassas, VA, USA), the RMA cell line RH30 (DSMZ, Braunschweig, Germany), and the human skeletal muscle cells (SkMC; Sigma Aldrich, Taufkirchen, Germany) were routinely cultured in DMEM medium (Biochrom, Berlin, Germany) supplemented with 10% fetal bovine serum (Biochrom, Berlin, Germany), 1% penicillin/streptomycin (Biochrom, Berlin, Germany) and 1% l-glutamine (Biochrom, Berlin, Germany) in a humidified atmosphere containing 5% CO₂ at 37 °C. Cell identity was proven by SLR analysis of the DNA profile using PowerPlex 16 (Promega, Mannheim, Germany). All cells were tested to be mycoplasma negative.

Namalwa (DSMZ #ACC24), JeKo-1 (DSMZ #ACC553) and BCWM.1 (kindly provided by S.P. Treon) were cultured in IMDM supplemented with 10% fetal bovine serum (HyClone), glutamine, and penicillin/streptomycin. HEK-293T/17 (ATCC #CRL11268) was cultured in DMEM supplemented with 10% fetal bovine serum, glutamine and penicillin/streptomycin. Authentication of the cell lines and their derived Cas9 clones was performed by STR profiling (Powerplex 16, Promega) followed by DSMZ’s online STR matching analysis. Cell cultures were regularly tested negative for mycoplasm contamination^{42 (link)}. Cas9-expressing cells are readily available upon request.

TF-1a cells were from The European Collection of Animal Cell Culture (Salisbury, UK) and were cultured in RPMI-1640 with 10% FCS and glutamine. Following treatments, cells were counted flow cytometrically using an internal standard as described [43 (link)]. Testing to authenticate the cell line was performed at the last passage of each thawed batch using multiplex short tandem repeat analysis (Powerplex 16, Promega, Southampton, UK). Mycoplasma testing was carried out routinely using the Mycoalert mycoplasma detection kit (Lonza, Rockland, USA) and following the manufacturer’s instructions.

All GBM PDCLs were established by the GlioTEx team (Glioblastoma and Experimental Therapeutics) in the Paris Brain Institute (ICM) laboratory and maintained at 37 °C, 5% CO₂ under neurosphere growth conditions using DMEM/F12 (Gibco, Life Technologies, Saint-Aubin, France) culture medium supplemented with 1% penicillin/streptomycin, B27 diluted 1:50 (Gibco), EGF (20 ng/mL), and FGF (20 ng/mL) (Preprotech, Neuilly-sur-Seine, France). The identity of all cell lines established at the ICM was confirmed by short tandem repeat (STR) assay according to the manufacturer’s instructions (PowerPlex 16, Promega, Charbonnières-les-Bains, France). PCR products were sent to Genoscreen (Lille, France) to determine STR profiles. The profiles were compared to the parental tumors and validated within three months of their use for the studies presented here.