GSM4104568: HeLa_tRNA_1; Homo sapiens; ncRNA-Seq
Media was aspirated and Trizol was added immediately to culture dishes. Total RNA was isolated using Trizol manufacturer's instructions (ThermoFisher) followed by treatment with Turbo Dnase (ThermoFisher). tRNA-sequencing: 1 micrograms of total RNA was ligated to annealed adapters under conditions as in (Shigematsu et al., 2017; PMID: 28108659) using 10-fold more RNA ligase. cDNA was synthesized using SuperScript IV Reverse Transcriptase (ThermoFisher 18090010) using manufacturer's recommended conditions and gel purified with denaturing polyacrylamide gel electorphoresis. Gel purified cDNA was then ligated using CircLigase (Lucigen CL4111K) with manufacturer's recommended conditions and libraries were amplified using Q5 DNA polymerase (New England Biolabs M0491) for 7-8 cycles then purified from a 2% agarose gel. Libraries were sequenced as single-end reads on an Illumina NextSeq 550.