ONT RNA-seq of the normal liver tissue (IBMC), Oxford Nanopore MinION

Total RNA was isolated and characterized as described above. The extraction of mRNA from the total RNA preparations was conducted using the magnetic beads based Dynabeads mRNA Purification Kit (Thermo Fisher Scientific, USA) for isolating polyA+ RNA, following the manufacturers recommendations. The mRNA preparations were immediately frozen and stored at -80C until use for nanopore sequencing. Nanopore sequencing was carried out on a MinION sequencer (ONT, Great Britain), using FLO-MIN106 flow cells with R9.4 chemistry and the Direct RNA sequencing kit (SQK-RNA002, ONT). The sequencing libraries were prepared strictly following the manufacturers protocol with a single exception - 750 ng of mRNA (poly+ RNA) instead of the recommended 500 ng. The SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) was used for reverse transcription and NEBNext Quick Ligation Module (New England Laboratories, Great Britain) for the end repair and ligation. The Agencourt RNAClean XP magnetic beads (Beckman Coulter, USA) were employed for nucleic acid purification. The mRNA was sequenced in a 72 h single run.