Optimizing the chemistry of charge tRNA-Seq. (A) Time required to complete periodate oxidation of the E.coli tRNA-Lys-CCA oligo on ice. Following oxidation, RNA was processed similar to Evans et al. (2017) to cleave off the 3′ adenosine. Successful cleavage produce E.coli tRNA-Lys-CC. CCA, input oligo. CC, product oligo. Mix, 50/50 mix of CCA and CC. SE, short exposure. LE, long exposure. (B) Time required to complete lysine cleavage of the E.coli tRNA-Lys-CCA oligo (CCA) at 45°C, pH=8. Cleavage at time 0 is likely due to the heat denaturation step performed in RNA loading buffer prior to running the gel. (C) TapeStation electropherogram comparing stability of whole cell RNA before and after 4 h lysine cleavage at pH=8 or 1.5 h borax cleavage at pH=9.5. tRNA range marked by red background, 18/28S by blue. See Figure 2—figure Supplement 1, panel B for RNA stability timecourse as it occurs on a gel. (D) Effect of individual components on cleavage of the E.coli tRNA-Lys-CCA oligo (CCA). All samples were processed as a one-pot reaction, except the borax sample which was processed similar to Evans et al. (2017). rSAP, shrimp alkaline phosphatase. (E) Ligation test comparing the effect of RNA processing. Deacylated and gel purified human tRNA was processed identically as in panel (D), then ligated to adapter l1Sp. Other adapters were tested with similar results (Figure 2—figure Supplement 5, panel A). (F) Baseline tRNA aminoacylation charge in H1299 cells grown in DMEM (4 replicates, bootstrapped 95% confidence interval of the mean). Charge on tRNAHis is possibly erroneously low because the discriminator base is shielded by base pairing (Heinemann et al., 2012), creating a steric hindrance for the splint assisted ligation.
Figure 2—figure supplement 1. Optimizing lysine induced cleavage.
Figure 2—figure supplement 2. Measurement bias in charge tRNA-Seq using blunt-end ligation.
Figure 2—figure supplement 3. tRNA-adapter blunt-end ligation attempted optimization
Figure 2—figure supplement 4. Splint assisted ligation is highly efficient.
Figure 2—figure supplement 5. Ligation tests, related to panel E.
Figure 2—figure supplement 6. RT readthrough comparing TGIRT to Maxima.
Figure 2—figure supplement 7. Sequenced controls.
Figure 2—figure supplement 8. tRNA homology requires careful PCR conditions.