MinION sequencing
RNA from whole tissue samples and cultured cells was isolated using TRI Reagent (Sigma) using manufacturer's protocol. Tissues were homogenized in TRI Reagent using glass Dounce homogenizer and cell monolayers were rinsed with TRI Reagent and lysed cells were gathered for further isolation according to manufacturer's protocol. DNA was removed from final sample by DNase treatment using TURBO DNA-free Kit (Thermo Fisher Scientific, Cat# AM1907) according to manufacturer's protocol. DNA-free RNA was stored in –80 °C. Libraries for direct RNA sequencing (DRS) were prepared using Direct RNA sequencing kit (ONT, Cat# SQK-RNA002) following manufacturers protocol, with adjustment to magnetic beads clean-up steps: each time KAPA Pure Beads (Roche) were used and RNA-bead mixture was incubated stationary on bench at RT. To improve sequencing performance and efficiency 90-150 ng of Saccharomyces cerevisiae or Saccharomyces pombe oligo(dT)-enriched mRNA was added to all samples. Sequencing experiments were performed on MinION device and Flow Cell Type R9.4.1 (ONT, Cat# FLO-MIN106D)