MinION sequencing
Total RNA was isolated with TRIzol (Thermo Fisher Scientific) according to the manufacturer's instructions, dissolved in nuclease-free water and stored at -80°C. The cap-enriched mRNA was prepared from 100 µg of total RNA with GST-eIF4EK119A protein and glutathione sepharose 4B (GE Healthcare), as described previously (Bilska et al., 2020). Nanopore Direct RNA libraries were prepared using Direct RNA Sequencing KIT SQK-RNA002 (ONT) according to the manufacturer's instructions, from 5 ug of human cap-enriched mRNA and, for better sequencing efficiency, mixed with the 100-150 ng of Saccharomyces cerevisiae oligo(dT)-enriched mRNA. Sequencing was performed with the MinION device and basecalled using Guppy.