RNA-Seq of rattus norvegicus: tenocytes under cyclic loading
Total RNA (650 ng for each sample) was first subjected to reverse transcription to generate cDNA. After reverse transcription, cDNA was PCR-amplified to obtain sufficient input material for sequencing. The resulting cDNA underwent end-repair and dA-tailing to create blunt, adenylated ends suitable for adapter ligation. ONT adapters (barcodes) were then ligated to the cDNA for each sample specifically. In the end, a bead-based cleanup was performed to remove enzymes, salts, primers, and short cDNA fragments, ensuring library purity and optimal sequencing performance. Finally, prepared libraries were quantified using Qubit flex fluorometer (Thermo Fisher Scientific, USA), and their integrity was evaluated using an Agilent Tapestation 4150 (Agilent Technologies, USA).50 fmol of cDNA was pooled and loaded onto a pre-conditioned R10.4.1 flow cell (Oxford Nanopore Technologies, Oxford, United Kingdom), primed with ONT sequencing buffer and loading beads, before sequencing on MinION. The sequencing run was initiated using MinKNOW software v24.11.10 (Oxford Nanopore Technologies, United Kingdom), with real-time basecalling performed using the Guppy (Dorado v7.6.8) basecaller.