GSM8895970: HEP3B, K290A, Rep 1; Homo sapiens; OTHER
Total RNA isolation was performed using Trizol Reagent (ThermoFisher Scientific) according to the manufacturer's protocol. Total RNA was then DNase treated with DNase I (NEB) and reconstituted in nuclease-free water. Briefly, total RNA of 1 µg was ligated to 18S rRNA custom-ordered reverse transcription adapter with T4 DNA Ligase (New England Biolabs) at room temperature for 10 min. Ligated RNA was reverse transcribed with Induro® Reverse Transcriptase (New England Biolabs) at 60°C for 30 min, then inactivated at 70°C for 10 min. The mixture was cleaned using 1.8-fold CleanNGS (CleanNA) according to the manufacturer's protocol. Reverse transcribed RNA (RT-RNA) was then ligated to an RNA Ligation Adapter (RLA) from the Direct RNA sequencing Kit (Oxford Nanopore, SQK-RNA004) at room temperature for 10 min, then purified with 0.4-fold CleanNGS (CleanNA). RNA library of 12 µl with 37.5 µl sequencing buffer and 25.5 µl library solution making 75 µl of final RNA library.