title: GSM6287512: wt_48h_3; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: All cells were grown in YPD media (2% peptone, 1% yeast extract, 2% glucose) at 30°C with shaking at 200 rpm. Media components were purchased from Formedium and media was sterilised by filtration. MEP experiments were performed as described with modifications (Cruz et al., 2018): cells were inoculated in 4 ml YPx (where x is the sugar) and grown for 6-8 hours then diluted in 25 ml YPD and grown for 16-18 hours to 0.2-0.6x107 cells/ml in 50 ml Erlenmeyer flasks. 0.125x107 cells per aged culture were harvested by centrifugation (15 s at 13,000 g), washed twice with 125 µl PBS and re-suspended in 125 µl of PBS containing ~3 mg/ml Biotin-NHS (Pierce 10538723). Cells were incubated for 30 min on a wheel at room temperature, washed once with 125 µl PBS and re-suspended in 125 µl YPD then inoculated in 125 ml YPD at 1x104 cells/ml in a 250 ml Erlenmeyer flask (FisherBrand FB33132) sealed with Parafilm. 1 µM β-estradiol (from stock of 1 mM Sigma E2758 in ethanol) was added after 2 hours. An additional 0.125x107 cells were harvested from the log phase culture while biotin labelling reactions were incubating at room temperature. Cells were harvested by centrifugation for 1 min, 4600 rpm, immediately fixed by resuspension in 70% ethanol and stored at -80°C. Percoll gradients (1-2 per sample depending on harvest density) were formed by vortexing 1 ml Percoll (Sigma P1644) with 110 µl 10x PBS in 2 ml tubes and centrifuging 15 min at 15,000 g, 4 °C. Ethanol fixed cells were defrosted and washed once with 1 volume of cold PBSE (PBS + 2 mM EDTA) before resuspension in ~100 µl cold PBSE per gradient and layering on top of the pre-formed gradients. Gradients were centrifuged for 4 min at 2,000 g, 4 °C, then the upper phase and brown layer of cell debris removed and discarded. 1 ml PBSE was added, mixed by inversion and centrifuged 1 min at 2,000 g, 4 °C to pellet the cells, which were then re-suspended in 1 ml PBSE per time point (re-uniting samples where split across two gradients). 25 µl Streptavidin microbeads (Miltenyi Biotech 1010007) were added and cells incubated for 5 min on a wheel at room temperature. Meanwhile, 1 LS column per sample (Miltenyi Biotech 1050236) was loaded on a QuadroMACS magnet and equilibrated with cold PBSE in 4 °C room. Cells were loaded on columns and allowed to flow through under gravity, washed with 1 ml cold PBSE and eluted with 1 ml PBSE using plunger. Cells were re-loaded on the same columns after re-equilibration with ~500 µl PBSE, washed and re-eluted, and this process repeated for a total of three successive purifications. After addition of Triton X-100 to 0.01% to aid pelleting, cells were split into 2 fractions in 1.5 ml tubes, pelleted 30 s at 20,000 g, 4 °C, frozen on N2 and stored at -70 °C. 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).