GSM8490653: tRNA_seq_2hpi_rep2; Acanthamoeba castellanii; ncRNA-Seq
The cell pellets were resuspended with 600 µl lysis buffer (20mM Tris pH 7.5, 150mM NaCl, 5mM MgCl2, 1mM dithiothreitol, 1% Triton X-100, 100 µg/ml of cycloheximide, and 100 µg/ml chloramphenicol), treated with 5 μl of TURBO DNase (2 U/μl, ThermoFisher Scientific) on ice for 10 min, and then centrifuged at 20,000 × g, at 4°C for 10 min. The tRNA fraction was collected by mirVana miRNA Isolation Kit (Thermo Fisher Scientific) and deacylated, preadenylated linker ligation, reverse transcription, cDNA circularization, and PCR amplification. mim-tRNA-seq