title: GSM8427449: BG2_RPMI_FLUC_A; Nakaseomyces glabratus; RNA-Seq
library_construction_protocol: RNA was extracted using a modified Zymo kit protocol following bead-beating with zirconia beads. RNA extraction was performed using silica columns purification method using Zymo reagents. The yeast pellets were thawed briefly on ice, then transferred to a screw cap tube and 200 μL of zirconia beads were added. 400 μL of RNA binding buffer (Zymo) was added, and the mixture was kept on ice for 1 minute. The tubes were transferred to PreCellys homogenizer (Bertin), and lysed using the following protocol: 10 seconds vortexing, followed by 10 seconds of waiting repeated 3 times. The cells were then transferred to ice for 1 minute. Vortexing and incubation on ice were repeated a total of 6 times. The tubes were then centrifuged at 12,000 × g for 2 minutes. The supernatant was transferred to a Zymo Spin IIIC column (Zymo) and centrifuged at 12,000 × g for 1 minute. 400 μL of ethanol was added to the flow through, mixed, transferred to a Zymo Spin IICG column (Zymo) and centrifuged for 1 minute. The column was washed with DNA/RNA Prep buffer (Zymo) and centrifuged for 1 minute, and then washed with DNA/RNA Wash buffer (Zymo) and centrifuged for 1 minute twice. The column was then transferred to a clean 1.5 mL tube, and RNA was eluted by adding 30 μL of water and centrifuging at 10,000 × g for 1 minute. The concentration of the samples was measured using Denovix. The quality and integrity of RNA was assessed on Fragment Analyzer (Agilent) using High Sensitivity RNA Kit DNF-472-1000 (Agilent). RQN and 28S/18S ratio were used to determine the quality of the sample. Lexogen QuantSeq Rev: RNA sequencing libraries were prepared from 500 ng total RNA using QuantSeq 3' mRNA-Seq V2 Library Prep Kit REV (Lexogen)