GSM8490643: Ribo_seq_8hpi_rep1; Acanthamoeba castellanii; OTHER
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. RNase I treatment, ribosome pelleting, size selection, preadenylated linker ligation, rRNA depeletion with Ribo-Zero Gold (Human/Mouse/Rat) kit (Illumina), reverse transcription, cDNA circularization, and PCR amplification.