GSM8578211: ERL, DMSO - Replicate 2; Homo sapiens; OTHER
All breast cancer cell lines were obtained from the American Type Culture Collection (ATCC). The 4T1.2 cell line was kindly provided by Dr Norman Pouliot, Olivia Newton-John Cancer Research Institute, Australia. All breast cancer cell lines were cultured in DMEM media supplemented with 10% fetal bovine serum (FBS) and 100 nM Sodium Selenite. All cell lines were tested for Mycoplasma infection and authenticated using short tandem repeat (STR) profiling by scientific services at QIMR Berghofer Medical Research Institute. Auranofin was purchased from Cayman Chemicals (Cat #: 15316). Erlotinib (Cat #: S7786) and Osimertinib (Cat #: S7297) were purchased from Selleck Chemicals.  TRi-1 was purchased from MedChem Express (Cat #: HY-125006. We used the Human GeCKOv2A & 2B CRISPR knockout pooled library to identify genes responsible for erlotinib resistance in TNBC cells. The library was a gift from Feng Zhang (Addgene # 1000000049) We established a stable Cas9-expressing MDA-MB-231 cell line by lentiviral transduction of Cas9 coding sequence. We then transduced Cas9-expressing MDA-MB-231 cells with GeCKO v2A and B library which contains 123,411 unique sgRNA sequences targeting 19,052 human genes and 1864 miRNAs (6 sgRNAs per gene, 4 sgRNAs per miRNA, and 1000 non-targeting control sgRNAs) at an MOI 0.3 to ensure each cell represents one sgRNA. The transduced cells were then selected with 1 µg/mL of puromycin for 7 days to generate a mutant cell line. The mutant cells were then treated with vehicle (DMSO) and Erlotinib (10 µM) for 7 days, respectively. After 7 days of treatment, the residual cells were collected, and genomic DNA was isolated from each treatment group. The sgRNA sequences were amplified using 9 different sets of primers listed in supplementary Table S2. The amplified sgRNA sequences were subjected to parallel amplicon sequencing at Ramaciotti Centre for Genomics to identify sgRNAs lost in Erlotinib-treated cells.