Telomerase activity assay

The telomerase assay was as previously described. Reactions contained 1x human telomerase buffer, 5 nM of 32P-end-labeled primer and 50 μM dNTP or rNTP mix as indicated in the figure legends. Each reaction was performed with four biological replicates. The reactions were started by the addition of 3 μL of immunopurified telomerase eluent, incubated at 37°C for a specified time course, then terminated with 2 μL of 0.5 mM EDTA and heat inactivated at 65°C for 20 min. An equal volume of loading buffer (94% formamide, 0.1 × Tris borate-EDTA [TBE], 0.1% bromophenol blue, 0.1% xylene cyanol) was added to the reaction eluent from the G-25 spin column. The samples were heat denatured for 10 min at 100°C and loaded onto a 14% denaturing acrylamide gel (7M urea, 1x TBE) and electrophoresed for 90 min at constant 38W. Samples were imaged using a Typhoon phosphorimager (GE Healthcare). Percent primer extension was quantitated using ImageQuant (RRID:SCR_014246).