GSM6592063: BY UBR1 wild-type 1; Saccharomyces cerevisiae; RNA-Seq
Total RNA was extracted from frozen cell pellets using the ZR Fungal/Bacterial miniprep kit (Zymo), according to the manufacturer's instructions. Briefly, total RNA was isolated from cell pellets in two batches, each containing equal numbers of BY UBR1 wild-type and BY UBR1 -469A>T samples. After thawing, pellets were resuspended in lysis buffer and transferred to screwcap lysis tubes containing glass beads. Tubes were secured in a Mini-BeadBeater (BioSpec Products, Bartlesville, OK, USA) and cells were processed in 5 cycles of 2 minutes of agitation followed by 2 minutes at −80 °C. The cell lysate/bead mixture was centrifuged for 1 minute at 16, 000 x g and 400 µL of 95% ethanol was added to the cleared supernatant followed by mixing. Samples were then spun through a binding column and on-column DNA digest was performed with DNase I (Zymo) according to the manufacturer's instructions. Total RNA was eluted from columns using 50 µL of RNase-free ultrapure water. The concentration of each sample was quantified using RiboGreen; all samples had a concentration greater than 300 ng/µL. The integrity of each sample was assessed at UMGC using the Tapestation (Agilent) and an RNA ScreenTape. RNA integrity numbers ranged from 9.7 to 10.0 (where 10.0 is the maximum possible score), with a median value of 9.9. All RNA samples were stored at −80 °C. We isolated mRNA from each total RNA sample using the 550 ng of total RNA input and the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). All samples were processed in a single batch and the isolated mRNA from each sample was used to prepare RNA sequencing libraries using the NEBNext Ultra II Directional RNA Library Prep kit (NEB) according to the manufacturer's instructions. Libraries were amplified using NEBNext Ultra II Q5 polymerase and unique combinations of primers from the NEBNext Multiplex Oligos for Illumina (NEB). The following amplification protocol was used: initial denaturation at 98 °C for 30 seconds, followed by 10 cycles of 98 °C (10 seconds; denaturation), 65 °C (75 seconds; annealing and extension), and a 65 °C final extension for 5 minutes. PCR reactions were pooled using equal amounts of DNA and submitted to UMGC for three quality control assays, which measured the library concentration by PicoGreen, library functionality by KAPA qPCR, and library size using the Tapestation electrophoresis system (Agilent). The resulting library contained a small amount of adapter dimer (approximately 9%), which was subsequently removed via a bead-based cleanup. The final, cleaned library passed all three QC assays and was sequenced on a Next-Seq 2000 instrument (Illumina) in paired-end mode with 150 bp reads. The sequencing run generated 1,367,252,076 reads with an average of 136,725,207 (range: 112,285,619 to 152,571,763) reads per sample.