title: Amplicon-Screening of genomic DNA regions of mutants in the Exon5 R175 position of TP53 (Exons 5, 6, 7, 8) in HCT116 cell line clones generated via CRISPR editing.
design: Amplicon-Screening of genomic DNA regions of mutants in the Exon5 R175 position of TP53 (Exons 5, 6, 7, 8) in HCT116 cell line clones generated via CRISPR editing.
library_construction_protocol: For the generation of R175-libraries, 4 x 106 HCT116 LSL/ΔIn5 cells were transfected with 6.25 µg pX459_blast_In5LSL and 6.25 µg of the HR700 vector library, respectively, using Lipofectamine 2000. For the generation of R175-libraries in H460 cells, 4 x 106 H460 LSL/Δ/Δ cells were transfected with 20 µg pX459_blast_In5LSL and 20 µg HR700 vector library using the Neon Transfection System (Thermo Fisher Scientific, MPK10025). Transfected cells were selected for 3 days with 20 µg/ml blasticidin and for 7 days with 1 µg/ml puromycin. 8 x 106 cells were then infected with AV-Cre and, after 5 days, cells were subjected to the different treatment regimens. Cells were treated with 10 µM N3a (BOC Sciences, B0084-425358), 75 nM RG7388 (MedChemExpress, HY-15676), 10 µM RO-5963 (Calbiochem, 444153), 1 µM MI-773 (Selleckchem, S7649), 750 nM AMG 232 (MedChemExpress, HY-12296) or the respective volume of DMSO (Carl Roth GmbH, 4720) as solvent control for 8 days. For irradiation experiments, an X-RAD 320iX tube was used with settings of 320 kV voltage and a current of 8 mA, with a dose rate ~1 Gy/min. Cells were further cultivated for 8 days after irradiation. 5-Fluorouracil (pharmacy of the Universitätsklinikum Gießen and Marburg) was administered at a concentration 5 µM for 24 h or 48 h, and cells were further cultivated for 8 days after treatment. For mutant p53 reactivation studies, cell libraries were treated with either 12.5 µM or 25 µM APR-246 (Sigma-Aldrich, SML1789) or 0.01 µM or 0.04 µM ZMC-1 (Abcam, NSC319726, #A24132) alone or in combination with 10 µM N3a or DMSO for a total of 8 days. Starvation experiments were performed to investigate the effect of nutrient deprivation on cell growth. Three different conditions were used to induce starvation: HBSS (Gibco, 14025050) for 3 days, DMEM without glucose (Gibco, 14025050) for 1 day, and DMEM without glutamine (Gibco, 21969) for 7 days. Following starvation, cells were allowed to recover and expand for either 1 day in the case of the -glucose condition or 7 days in the case of the HBSS or -glutamine conditions. Genomic DNA of mutant cells was isolated using DNA Blood Mini Kit (QIAGEN, 51106) following the manufacturer's protocol. a nested PCR strategy was used to selectively amplify either edited or edited and recombined alleles. The input amount of genomic DNA and number of PCR reactions were adjusted to achieve a minimum average coverage of 500 cells/variant. The PCR products were pooled, purified, and diluted 1:1000 for the second step. Editing-specific primers were used for the second step PCR, exon-wise. The PCRs were performed with Q5 High-Fidelity DNA Polymerase (New England Biolabs, M0491) following the manufacturer's protocol. PCR products were purified using PCR purification kit according to the manufacturer's protocol. PCR amplicons were purified with AMPure XP beads (Beckman Coulter, A63880) and sequencing libraries were prepared from 5 ng of the purified amplicon using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, E7370L) according to the manufacturer's protocol. The quality of sequencing libraries was validated with a Bioanalyzer 2100 using the Agilent High Sensitivity DNA Kit (Agilent, 5067-4626).