MinION sequencing
Total RNA was isolated from cells with TRIzol reagent (Thermo Fisher Scientific), according to the manufacturer'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). Direct RNA sequencing was performed as described previously (Bilska et al., 2020). 3.5-5 μg of total mRNA was mixed with 50-200 ng oligo-(dT)25-enriched mRNA from Saccharomyces cerevisiae yeast to optimize sequencing efficiency, and standards with predefined poly(A) lengths, and processed with a Direct RNA Sequencing Kit (catalog no. SQK-RNA002, Oxford Nanopore Technologies) according to the manufacturer's instructions . Sequencing was performed with MinION device (Oxford Nanopore Technologies, Flow cell type FLO-MIN106; RevD).