Generating Fully Q-modified Cells

HEK293T, HeLa, and MCF7 cells were cultured with complete DMEM or EMEM medium under standard conditions. The 0Q and 100Q cells were obtained as previously described (Wang et al. 2018 (link)). Briefly, MCF7 cells were grown in EMEM medium (ATCC, 30-2003) with dialyzed 10% FBS (Thermo Fisher Scientific 26400044), 0.01 mg/mL bovine insulin (Sigma-Aldrich I0516), and 10 nM β-estradiol (Sigma-Aldrich E2758) to 80% confluency and passaged. HEK293T and HeLa cells were grown in Hyclone DMEM medium (GE Healthcare Life Sciences SH30022.01) with 10% dialyzed FBS to 80% confluency and passaged. Total RNAs were extracted using TRIzol (Thermo Fisher Scientific 15596026) by following the manufacturer's manual at each passage and the Q-modification fraction of the tRNA^His/Asn was determined using APB gel and northern blot. After ∼10 passages, Q was depleted from these three cell lines (0Q cells). To get 100Q cells (100% Q-modified), 0Q cells were grown to 60%–80% confluency and queuine was added to the medium to 1 µM final concentration. The cells were cultured for an additional 24 h to become fully Q-modified.