The WT and mutant full-length human POT1 genes were cloned in the pLU-EF1A-iBlast (pLU) vector with an N-terminal 1xFlag tag and 4 μg of vector was used to transfect HEK 293T cells using lipofectomine 2000 (Invitrogen) to test expression. HEK293T cells were cultured in growth medium containing Dulbecco's modified Eagle medium (Cellgro) supplemented with 10% fetal bovine serum (Sigma) and 100 units ml−1 penicillin and 100 μg ml−1 streptomycin (Sigma). All human cells were cultured at 37 °C with 5% CO2 and harvested 48 h after transfection.
Stable HEK293T cell lines overexpressing 1xFlag-POT1 were carried out using lentiviral infection to deliver WT and mutant human POT1 genes in the pLU vector with blasticidin resistance. A pLKO.1 vector with puromycin resistance carrying the shRNA (TRCN0000009837) targeting the 3′UTR of endogenous POT1 (shPOT1) was obtained from the Sigma Mission shRNA library and was delivered with lentiviral infection. Lentiviral particles were prepared by lipofectomine 2000 (Invitrogen) transfection of HEK293T cells with the pLU or pLKO.1 and lentiviral production vectors. Growth media was spiked with 5 μg ml−1 blasticidin S, and 2 μg ml−1 puromycin.
Stable HEK293T cell lines overexpressing 1xFlag-POT1 were carried out using lentiviral infection to deliver WT and mutant human POT1 genes in the pLU vector with blasticidin resistance. A pLKO.1 vector with puromycin resistance carrying the shRNA (TRCN0000009837) targeting the 3′UTR of endogenous POT1 (shPOT1) was obtained from the Sigma Mission shRNA library and was delivered with lentiviral infection. Lentiviral particles were prepared by lipofectomine 2000 (Invitrogen) transfection of HEK293T cells with the pLU or pLKO.1 and lentiviral production vectors. Growth media was spiked with 5 μg ml−1 blasticidin S, and 2 μg ml−1 puromycin.