Purification of tRNAs and reverse-phase HPLC analysis of nucleosides

Bulk tRNA was purified as described [¹¹]. Specific true native tRNAs and tRNAs overexpressed from pBSKrna-derived plasmids were purified from bulk tRNA by the Chaplet Column Chromatography method using biotinylated DNA probes immobilized on a HiTrap Streptavidin HP column [⁹,²⁵]. The probes were complementary to the specific sequence of each tRNA (Supplementary Table S2). Analysis of nucleosides by reverse-phase HPLC was performed as described [¹¹]. The nucleosides were identified according to their UV spectra, relative retention times, and by comparison with appropriate controls, including synthetic markers [⁷,⁹,²⁶].