Fig. 1

Direct cDNA sequencing workflow. The workflow comprises four main steps that include sample preparation, library preparation, sequencing, and computational data analysis. Sample preparation includes bacterial growth, cell lysis, total RNA extraction, ribosomal RNA depletion (16S and 23S), and in vitro polyadenylation to ensure compatibility with the Oxford Nanopore cDNA protocol. Library preparation involves reverse transcription with strand switching, RNA degradation, second-strand synthesis, end-repair, native barcoding, and adapter ligation. Sequencing is performed on a MinION device after flow cell priming and setup in MinKNOW, enabling real-time data acquisition. Computational analysis comprises basecalling, alignment to a reference genome, quantification of gene expression, principal component analysis (PCA), differential expression analysis (DESeq2), gene set enrichment analysis (GSEA), and operon identification. Figure created with BioRender (https://BioRender.com/k62c145).