Deep sequencing of tRNAs have historically been nutoriously difficult. Here, we benchmark a newly developed library prep protocol termed OTTR agains intact tRNAs as well as tRNA fragments and show that OTTR outperforms any other commercial cloning protocol. Overall design: Intact tRNA from mouse testisand S. cerevisiae, as well as tRNA fragments (tRFs) from mouse cauda epididymis and S. cerevisiae were cloned using three different protocols. Results were then compared to published data generated using different clonign protocols.