Small RNA sequencing of serum-derived RNA for the evaluation of 5'' tRNA halves depletion protocols

5'' tRNA halves are abundantly present in small RNA libraries prepared from serum samples, thereby limiting the detection of other small RNA species. In this study, we developed and compared two protocols for the selective depletion of 5'' tRNA halves in RNA isolated from murine serum samples. To evaluate the efficacy of both protocols on small RNA sequencing, we performed the two 5'' tRNA halves depletion protocols on total RNA isolated from a serum pool collected from healthy mice. Each RNA sample was divided into three and the resulting fractions were either depleted using beads or RNase H, or used as a control. The experiment was performed in triplicate. Upon depletion, libraries were prepared using TruSeq Small RNA Library Preparation kit. For both protocols, very high depletion efficiencies were observed, with more than 99% of the targeted tRNA-types being depleted. This resulted in a more than 6-fold increase in mapped miRNA reads and 60% more detected mature miRNAs when performing small RNA sequencing on murine serum samples. Importantly, we observed no considerable effects on the relative expression values of miRNAs. Overall design: Serum samples from healthy mice were combined together into a total of 3 serum pools. The RNA extracted from each pool was divided into three and the resulting fractions were either depleted using beads or RNase H, or used as a control.