MinION sequencing
Total RNA was isolated from cells with TRIzol reagent (Thermo Fisher Scientific), according to the manufacturerâ€™s instructions, dissolved in nuclease-free water and treated with TURBO DNase (Thermo Fisher Scientific; AM1907; or InvitrogenTM,AM2238). To assess the integrity of isolates, each RNA sample after DNAse treatment was analyzed with Agilent 2200 TapeStation system, using Agilent High Senitivity RNA ScreenTape (Agilent, 5067-5579). For library preparation 400ng of total RNA was used for library preparation using cDNA-PCR Sequencing Kit SQK-PCS111 kit or PCR-cDNA Barcoding Kit (SQK-PCB111.24) according to manufacturer's instruction. Libraries were amplified with 14 cycles of PCR reaction, DNA concentration was measures using Qubit 1xdsDNA high sensivity kit (Invitrogen, Q33231), and then if multiplexed, they were diluted and mixed equally to reach a final concentration of 60-100 fmol.