BioProject

Currently available methods do not sample the entire tRNA pool or lack specificity for tRNAs. More...

Currently 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’). Less...