Generating Fibroblasts and HEK293T Cells with POT1 Variants

Patient-derived skin fibroblasts were obtained from a skin punch biopsy and grown in NUT.MIX.F-12(HAM) w/Glutamax (HAMF-12), supplemented with 10% FBS and penicillin/streptomycin (15140122; Gibco). They were cultured in DMEM (10-013-CV; Corning) supplemented with 1× penicillin/streptomycin (15140122; Gibco) and 10% heat-inactivated FBS (S1620; Biowest) at 37°C and 5% CO₂. HEK293T were selected with shRNAs targeting POT1 as in our previous publication (Rice et al., 2017 (link)). These cells were then introduced with Flag-POT1(WT) or FlagPOT1(L259S) using pLU iBLAST vectors. Cells were selected using growth medium supplemented with 5 μg/ml blasticidin S and 2 μg/ml puromycin.