Nanopore-Based RNA Sequencing Workflow

The purification of mRNA from total RNA samples was achieved using the Dynabeads mRNA Purification kit (Thermo Fisher Scientific). The subsequent reverse transcription reaction was performed using SuperScript IV reverse transcriptase (Thermo Fisher Scientific). Preparation for RNA sequencing was carried out using the low-input PCR barcoding kit and the cDNA-PCR Sequencing kit (Oxford Nanopore Technologies), which 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). Only D²-Reads with a quality score above eight were used for further alignment. The quality score is measured based on the basecalling algorithm albacore (Nanopore), defined as −10 × log10 (probability of incorrect base call). Reads were rearranged in accordance to their barcode and trimmed using Porechop (https://github.com/rrwick/Porechop), with alignment performed using minimap2 (https://github.com/lh3/minimap2) and postprocessed by sam-tools. Mapped reads were normalized by DESeq (Love et al, 2014 (link)). The expression matrix was analyzed with AutoPIPE (https://github.com/heilandd/AutoPipe), a supervised machine-learning algorithm and visualized in a heat map as described previously (Henrik Heiland et al, 2019) (link).