Protocol detail

DT40 Cell Mutant Analysis for DNA Damage Response

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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.

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Zámborszky J., Szikriszt B., Gervai J.Z., Pipek O., Póti Á., Krzystanek M., Ribli D., Szalai-Gindl J.M., Csabai I., Szallasi Z., Swanton C., Richardson A.L, & Szüts D. (2016). Loss of BRCA1 or BRCA2 markedly increases the rate of base substitution mutagenesis and has distinct effects on genomic deletions. Oncogene, 36(6), 746-755.

Publication 2016

Gentra Puregene Cell Kit

Brca1 Brca2 Cell clones Cell lines Cells Chicken Fetal bovine serum Gene mutations Genomic Methylcellulose Sensitivity Serum

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Variable analysis

independent variables

MMS (Sigma-Aldrich, St Louis, MO, USA) or mock treatments

dependent variables

MMS sensitivity measured by counting surviving cell colonies after plating treated cells in medium containing 1% methylcellulose

control variables

Wild-type Clone18 DT40 cell line
BRCA1−/−, BRCA1+/− mutant DT40 cell lines
BRCA2−/−, BRCA2+/− (originally termed BRCA2-/con1) mutant DT40 cell lines
PCNAK164R/K164R (referred to in the text as PCNAK164R) DT40 cell line
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
One million cells were treated 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)

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