GSM6290746: wild type YPFructose 48h lane7129_CCAACA_E34_8_FRU_48h; Saccharomyces cerevisiae; RNA-Seq
Cells were re-suspended in 50 µl Lysis/Binding Buffer (from mirVANA kit, Life Technologies AM1560), and 50 µl 0.5 µm zirconium beads (Thistle Scientific 11079105Z) added. Cells were lysed with 5 cycles of 30 s 6500 ms-1 / 30 s ice in an MP Fastprep bead beater or for 3 min at power 12 in a Bullet Blender (ThermoFisher) in cold room, then 250 µl Lysis/Binding buffer added followed by 15 µl miRNA Homogenate Additive and cells were briefly vortexed before incubating for 10 minutes on ice. 300 µl acid phenol : chloroform was added, vortexed and centrifuged 5 min at 13,000 g, room temperature before extraction of the upper phase. 400 µl room temperature ethanol and 2 µl glycogen (Sigma G1767) were added and mixture incubated for 1 hour at -30 °C before centrifugation for 15 minutes at 20,000 g, 4 °C. The pellet was washed with cold 70% ethanol and re-suspended in 10 µl water. 1 µl RNA was glyoxylated and analysed on a BPTE mini-gel, and RNA was quantified using a PicoGreen RNA kit (Life Technologies R11490) or Qubit® RNA HS Assay Kit. 150 ng RNA was used to prepare libraries using the NEBNext Ultra II Directional mRNAseq kit with poly(A)+ purification module (NEB E7760, E7490) as described with modifications: Reagent volumes for elution from poly(T) beads, reverse transcription, second strand synthesis, tailing and adaptor ligation were reduced by 50%; libraries were amplified for 13 cycles using 2 µl each primer per 50 µl reaction before two rounds of AMPure bead purification at 0.9x and elution in 11 µl 0.1x TE prior to quality control using a Bioanalyzer HS DNA ChIP (Agilent) and quantification using a KAPA Library Quantification Kit (Roche).