MinION sequencing; Nanopore direct RNA sequencing of S. cerevisiae Mex67-AA (no rapamycin), W303, 25°C, not labelled with 4tU, technical replicate b
Biotinylation and purification of newly synthesized RNA were based on the protocol by Barrass et al.(Barrass and Beggs, 2019) with minor modifications. Briefly, 450 µg of total RNA extracted from 4tU-labelled samples dissolved in TE-buffer (10 mM Tris HCl pH 7.0, 1 mM EDTA) was spiked with 1% (w/w) of 4tU-labelled S. pombe total RNA, combined with EZ-link HPDP Biotin (final concentration 0.2 mg/ml), and incubated at 65˚C for 15 min. Unincorporated biotin was removed by desalting the samples in Zeba Spin Desalting Columns (7K MWCO). Biotinylated RNA was precipitated in 70% EtOH and 60 mM LiCl over-night at -20˚C, and the pellet was washed twice with 80% EtOH at 4˚C for 1h, and re-dissolved in 200 µl nuclease- ree water. Dynabeads MyOne Streptavidin C1 beads (100 µl slurry per sample) were washed once with bead buffer (100 mM Na-phosphate pH 6.8, 200 mM NaCl, 25 mM MgCl 2 , 0.1% SDS) in low DNA binding tubes. Washed beads were resuspended in 200 µl of bead buffer and blocked with 12.5 µg tRNA (Roche 28473522) and 200 µg glycogen by rotating the beads for 20 min at room temperature and washed once with bead buffer. Biotinylated RNA samples contained in bead buffer were added on the blocked beads and then rotated for 30 min at room temperature. Beads were washed four times with bead buffer before elution of 4tU RNA with 100 µl of 0.7M β-mercaptoethanol. Eluted RNA was precipitated over-night at -20˚C in the presence of 0.3M sodium acetate pH 5.3, 70% EtOH and 1 µg of glycogen. Pelleted RNA was washed three times with 70% EtOH and resuspended in 10 µl nuclease free water. The spike-in RNA was extracted from S. pombe cells grown exponentially in YPAD medium at 30 ˚C and labelled with 4tU (5 mM) for 10 min.  Nanopore direct-RNA libraries were prepared with Direct RNA Sequencing Kit (Oxford Nanopore Technologies, SQK-RNA002) using 150 ng of Saccharomyces cerevisiae oligo(dT)-enriched mRNA, mixed with RNA from other species (murine cells). Sequencing was performed with MinION device (Oxford Nanopore Technologies, Flow cell type FLO-MIN106; RevD) and basecalled with Guppy (Oxford Nanopore Technologies)