MinION sequencing; Nanopore direct RNA sequencing of S. cerevisiae WT, W303, 38°C 12min, control for mft1 series, biological replicate 1
RNA from yeast cells was extracted using the hot acid phenol method. Cell pellets from 10-30 ml cultures at OD 0.3 to 0.6 were resuspended in 400-600 μl of TES buffer (10 mM Tris [pH 7.5], 5 mM ethylenediaminetetraacetic acid, and 1% sodium dodecyl sulfate [SDS]) and complemented with an equal volume of acid phenol (catalog no. P4682, Sigma-Aldrich). The samples were incubated at 65°C with vortexing in two rounds for 40 and 25 min. Between and after incubations, the samples were centrifuged at 20,000 g for 10 min at 4°C. The resulting supernatant was vortexed with an equal volume of chloroform (catalog no. 112344306, Chempur) and centrifuged again. The RNAs from the supernatant were precipitated using 96% ethanol and a 65 mM final concentration of LiCl. Yeast polyadenylated RNA for DRS library construction and reverse-transcription qPCR analysis was extracted from 35-75 μg total RNA using the ThermoFisher dynabeads oligo dT(25) kit (catalog no. 61002 or 61005) according to the manufacturer's protocol. 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 (human cells). Sequencing was performed with MinION device (Oxford Nanopore Technologies, Flow cell type FLO-MIN106; RevD) and basecalled with Guppy (Oxford Nanopore Technologies)