title: RNA-Seq of HepG2 cell line - Oxford Nanopore MinION
design: Total RNA was isolated and characterized as described above (section 5.2). 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) were used as an input both in the case of samples from human liver and HepG2 cells, 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 from HepG2 was sequenced in a 72 h single run. The output was 0.75 Gb sequenced transcripts (0.766 million reads) with a median length of 1.56 kb. The mRNA from tissue liver of donor A was sequenced for 26 h. The flow cell was regenerated with the Flow Cell Wash Kit (ONT), strictly following the manufacturers guidance. Afterwards, the newly prepared sequencing library from liver mRNA of donor A was loaded on the flow cell and a 48 h sequencing run was initiated.