Chapter 5 —— 127 —— Characterization of genome editing events After sub-culturing selector AAV transduced cells for more than two weeks, reporter-positive cells were sorted by using either a BD FACSAria III flow cytometer (BD Biosciences) or a CytoFLEX SRT Cell Sorter (Beckman). Next, individual reporter-positive cells were seeded in wells of 96-well plates in 1:1 mixtures of culture medium and FBS supplemented with penicillin/streptomycin at 100 U ml-1 with or without 0.2 μM ouabain. Moreover, to increase cell cloning efficiencies, α-thioglycerol and bathocuprione disulphonate (both from Sigma-Aldrich) were added at final concentrations of 50 μM and 20 nM, respectively. Single cell-derived clones expressing reporter proteins were arbitrarily collected after 2-3 weeks for genotyping through junction PCR analyses using the Phire Tissue Direct PCR Master mix according to the manufacturer’s instructions (Thermo Fisher Scientific; Cat. No.: F170L). The PCR mixture reagents and thermocycling parameters used in the clonal screens are indicated in the Supplementary Tables S2 - S5, S10 and S11. Statistical analyses Statistical analyses were performed with the aid of the GraphPad Prism software (version 9.3.1) on datasets derived from a minimum of three biological replicates. Two-tailed unpaired Student's t tests were performed to assess statistical significance amongst two independent experimental groups. Details on statistical parameters are also indicated in the figure legends where applicable. P values inferior to 0.05 were considered to be statistically significant. Acknowledgements The authors are thankful to Thilo M. Buck and Jan Wijnholds (Department of Ophthalmology, Leiden University Medical Centre, The Netherlands) for their advice during the setting-up of AAV vector production procedures in our laboratory. The authors also thank the personnel of the Flow Cytometry Core Facility of the LUMC for aiding with the cell sorting procedures.
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