Bulk tRNA was prepared from fresh cells from 750
mL of cultures of the different S. pombe strains
grown in YPD (1% yeast extract, 2% peptone, 2% dextrose, BD Difco
YPD) or 0.5% yeast extract (Difco), 2% bactopeptone (Difco), and 3%
dextrose (Fluka) at 30 °C, as described previously.85 (link) tRNAAspGUC was extracted
from bulk tRNA using a biotinylated primer (5′biotin-GCAAGCGTGACAGGCTTG-3′,
Integrated DNA Technologies) bound to a HiTrap Streptavidin HP column
(1 mL, GE Healthcare Life Sciences).85 (link) Twenty-five
micrograms of tRNAAspGUC was digested to nucleosides
that were then separated by LC–MS.85 (link) To compare tRNA concentrations, we compared the ratio of the levels
of the modified bases Q (410 m/z) and m5C (258 m/z)
in each sample by integrating the peak area from the extracted ion
chromatograms. All tRNA extractions and analysis for Q content were
performed at least twice independently.
mL of cultures of the different S. pombe strains
grown in YPD (1% yeast extract, 2% peptone, 2% dextrose, BD Difco
YPD) or 0.5% yeast extract (Difco), 2% bactopeptone (Difco), and 3%
dextrose (Fluka) at 30 °C, as described previously.85 (link) tRNAAspGUC was extracted
from bulk tRNA using a biotinylated primer (5′biotin-GCAAGCGTGACAGGCTTG-3′,
Integrated DNA Technologies) bound to a HiTrap Streptavidin HP column
(1 mL, GE Healthcare Life Sciences).85 (link) Twenty-five
micrograms of tRNAAspGUC was digested to nucleosides
that were then separated by LC–MS.85 (link) To compare tRNA concentrations, we compared the ratio of the levels
of the modified bases Q (410 m/z) and m5C (258 m/z)
in each sample by integrating the peak area from the extracted ion
chromatograms. All tRNA extractions and analysis for Q content were
performed at least twice independently.
