MinION-based RNA Sequencing Workflow

The purification of mRNA from total RNA samples was achieved using the Dynabeads mRNA Purification Kit (Thermo Fisher Scientific, Carlsbad, CA, USA). The subsequent reverse transcription reaction was performed using SuperScript IV reverse transcriptase (Thermo Fisher Scientific). For preparation of RNA sequencing, the Low Input by PCR Barcoding Kit and the cDNA-PCR Sequencing Kit (Oxford Nanopore Technologies, Oxford, UK) were used as recommended by the manufacturer. RNA sequencing was performed using the MinION Sequencing Device, the SpotON Flow Cell and MinKNOW software (Oxford Nanopore Technologies) according to the manufacturer’s instructions. Base calling was performed by Albacore implemented in the nanopore software (Oxford Nanopore Technologies). Only D2-reads with a quality score above 8 were used for further alignment. Reads were re-arranged in accordance to their barcode and trimmed by Porechop (https://github.com/-rrwick/Porechop). Alignment was performed by minimap2 (https://github.com-/lh3/minimap2) and processed by sam-tools. Mapped reads were normalized by DESeq2 [32 (link)]. The expression matrix was analyzed with AutoPipe (https://github.com/heilandd/AutoPipe) by a supervised machine-learning algorithm and visualized in a heatmap. (Data available in GEO: in preparation).