GSM3938311: RPFA0002_23_2B_BY_OAF1_L63S; Saccharomyces cerevisiae; RNA-Seq
The plate was centrifuged for 3 min at 2,100g, the supernatant was removed, and each cell pellet was resuspended in 1 ml of H2O and transferred to a 1.5 ml centrifuge tube.  After centrifugation for 2 min at 16,000g, the supernatant was removed and the cell pellet was flash frozen by immersion of the tube in liquid nitrogen and stored at -80. Total RNA was isolated from cell pellets in 5 batches, with each batch containing a sample of each of the 5 strains, using the ZR Fungal/Bacterial RNA mini-prep kit, including the DNase I on-column digestion step (Zymo Research). Cell pellets chosen for RNA isolation had the most uniform OD600 reading across strains, with an average OD600=0.37 (range of 0.34 to 0.40). Each cell pellet was resuspended in lysis buffer and transferred to screw-capped tubes containing glass beads and the cells were broken open using a mini-beadbeater (Biospec Products) in 5 cycles of 2 min bead beating followed by 2 min at -80℃. The total RNA was eluted in 50 ul of DNAse/RNAse free water. Poly-A RNA was extracted from 550 ng of total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). PolyA RNA was used as input into the NEB Ultra II Directional RNA library kit for Illumina (NEB E7760) in two batches. NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set1) were used to amplify and barcode the libraries using the following cycling conditions: Initial Denaturation at 98℃ for 30s and 10 cycles of: 98℃ for 10 sec and 65℃ for 75 sec, followed by a 65℃ extension for 5 min. The amplified DNA was quantified using Qubit DNA HS. Sample concentrations ranged from 47-102 ng/ul. An equal concentration of each of the 25 barcoded libraries was pooled and the average fragment size of the library was determined using a Agilent High Sensitivity chip. High-output sequencing was performed on an Illumina NextSeq 550 at the University of Minnesota Genomics Core.