show Abstracthide AbstractCurrently available methods do not sample the entire tRNA pool or lack specificity for tRNAs. Combining and optimizing high-throughput sequencing methods, we developed Lotte-seq a highly specific protocol for efficient and comprehensive analysis of tRNAs. Ligation of a hairpin adapter with 3'-TGGN overhang enables highly specific selection of tRNAs with 3'-CCA end, rendering it a powerful tool to analyze the tRNA pool of cells under different conditions and for a variety of tRNA-related diseases. For validating Lotte-seq, tRNAs from HEK293T, S. oleracea, S. cerevisiae, D. discoideum, E. coli and G. stearothermophilus were sequenced using three different library preparation strategies (Standard Illumina sRNA TruSeq; Illumina sRNA TruSeq optimized for tRNAs and Lotte-seq). The purified library constructs were analyzed by a 2100 Bioanalyzer (Agilent) at the Max Planck Institute for evolutionary anthropology (MPI EVA Leipzig) for concentration and purity. High-throughput analysis of the libraries was done as single end run (150 nt) with a MiSeq System (Illumina®) at the MPI EVA (Leipzig) and a custom primer designed for Illumina MiSeq analysis (5'-CACTGTCGGTACCGAGCTTGCATGGAGTCCTA-3').