title: GSM8410970: DRS_W303_+_thiolutin_12min_25°C_repA; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: RNA extraction was performed using the hot acid phenol method. Cell pellets were resuspended in 400 μl phenol solution saturated with 0.1 M citrate at pH 4.3 (Sigma; P4682), followed by the addition of 400μl of TES buffer (10 mM Tris pH 7.5, 5 mM EDTA, 1 % SDS). Samples were vortexed for 45 min at 65 °C, then centrifuged at 4 °C for 10 min. The supernatant was transferred to a fresh tube, and 400 μl phenol solution saturated with 0.1 M citrate at pH 4.3 was added. The samples were again vortexed for 20 min at 65 °C and then centrifuged at 4 °C for 10 min. The supernatant was transferred to a fresh tube and 400 μl of chloroform was added (C2432; Sigma). The samples were briefly vortexed at room temperature to remove phenol, and centrifuged at 4 °C for 10 min. The supernatant was transferred to a fresh tube; 45 μl of 2 M LiCl was added and 1 ml of 95 % ethanol. Samples were precipitated at – 80 °C for at least 30 min, then centrifuged at 4 °C for 25 min, washed with 400 μl of 80 % ethanol, and dried at 37 °C after removing the supernatant. Pellets were resuspended in nuclease-free water, and RNA concentration was measured using a Nanodrop apparatus. The pA+ fraction was prepared using magnetic beads coupled to oligo-dT from LifeTechnologies (61005). 35 μg of total RNA was resuspended in 50 μl of nuclease-free water. The RNA was mixed with 50 μl of binding buffer (20 mM Tris-HCl, ph 7.5, 1 M LiCl, 2 mM EDTA) and denatured for 2 min at 65 °C before cooling on ice. 100 μl of slurry beads per 35 μg of total RNA was pre-washed 3 times in 1 ml of binding buffer and resuspended in 50 μl of binding buffer per sample. The beads were added to the denatured RNA and incubated at room temperature with occasional shaking for 20 min. The supernatant was removed, and beads were washed twice with 100 μl of wash buffer (10 mM Tris-HCl pH 7.5, 150 mM LiCl, 1 mM EDTA) and after removing any remnants of wash buffer, resuspended in 12 μl of nuclease-free water. The beads were then incubated for 2 min at 80 °C, and the supernatant removed from the beads was utilized for sequencing library preparation and/or qPCR analyses as the pA+ fraction. Sequencing libraries were prepared using the Direct RNA Sequencing Kit (Oxford Nanopore Technologies, SQK- RNA002) according to the manufacturer's protocol, using 50–200 ng oligo-dT(25)-enriched mRNA from Saccharomyces cerevisiae yeast mixed with cap-enriched or total RNA from other organisms (human, mouse, A. thaliana, or C. elegans) RNA-seq (Nanopore Direct RNA Sequencing)