Gentra puregene cell kit

DT40 wild-type cell line stocks originally sourced from the Institute of Animal Health (now Pirbright Institute, UK) were obtained from the laboratory of Dr Julian E. Sale, MRC Laboratory of Molecular Biology, Cambridge, UK. Cells were grown at 37° under 5% CO₂ in RPMI-1640 medium supplemented with 7% fetal bovine serum, 3% chicken serum, 50 μM 2-mercaptoethanol, and penicillin/streptomycin. Single cell clones were isolated and grown prior to sample preparation. Genomic DNA for both SNP array analysis and DNA sequencing was prepared using the Gentra Puregene Cell Kit (Qiagen). The sample preparation for the SNP array analysis took place previously, and the cell clones were frozen in 90% fetal bovine serum plus 10% DMSO, stored in liquid nitrogen, and re-thawed prior to the preparation of the DNA sequencing sample.

The following DT40 cell lines were used: wild-type Clone18,^{52 (link)}BRCA1^−/− and BRCA1^+/− mutants,^{19 (link)}BRCA2^−/− and BRCA2^+/− (originally termed BRCA2^-/con1),^{20 (link)}PCNA^K164R/K164R (referred to in the text as PCNA^K164R).^{33 (link)} All gene mutations were authenticated using the whole-genome sequence data. Cells were grown at 37 °C under 5% CO₂ in Roswell Park Memorial Institute-1640 medium supplemented with 7% fetal bovine serum and 3% chicken serum. MMS (Sigma-Aldrich, St Louis, MO, USA) or mock treatments were performed on one million cells for 1 h. Single cell clones were isolated and expanded to two million cells prior to genomic DNA preparation using the Gentra Puregene Cell Kit (Qiagen, Hilden, Germany). MMS sensitivity was measured by counting surviving cell colonies after plating treated cells in medium containing 1% methylcellulose.

Cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen #11965) containing 10% fetal bovine serum (FBS, Sigma), 1% NEAA (Invitrogen 15140122) and 7 μL/mL penicillin-streptomycin (Invitrogen 15140122) in a humidified atmosphere of 5% CO₂ at 37°C, as described [17 (link)]. Cells were tested for mycoplasma contamination using the MycoAlert detection kit (Lonza) and genotyped, as described [18 (link)]. UM-SCC lines were transduced with the Human Genome-wide CRISPR KnockOut (GeCKO) pooled library, version 2A which was a gift from Feng Zhang [15 (link)]. Conditions for transduction were established for a multiplicity of infection (MOI) of 30%. After 7 days of puromycin selection, the cells were expanded and seeded per treatment. To preserve at least 300x coverage, 30 million cells were seeded per treatment for the GeCKO libraries. At the end of treatment, DNA was extracted from the remaining cells using Gentra Puregene Cell Kit (Qiagen), as described [19 (link)]. For cisplatin treatment, cells were dosed with 0.125 μM cisplatin (Selleckchem S1166) or DMSO (Sigma Aldrich) for 24 hours, once a week for two weeks.

DNA extraction from PNT1A and LNCaP cells and clinical samples was carried out using the Gentra Puregene Cell Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. To evaluate the methylation pattern, 1 µg of genomic DNA was modified using the EZ DNA Methylation-Gold kit (ZYMO RESEARCH, Irvine, CA, USA) according to the manufacturer’s instructions. The modified DNA was stored at −80 °C.