GSM2877787: wt_48h_YPD_5; Saccharomyces cerevisiae; RNA-Seq
Percoll gradients (1 for 7.5 hr or 2 for 24/48 hr time points) were formed by vortexing 1ml Percoll (Sigma P1644) with 110µl 10x PBS in 2ml tubes and centrifuging 15 min at 15,000 g, 4 °C. Ethanol fixed cells were defrosted and washed 1x with 1 volume of cold PBSE (PBS + 2 mM EDTA) before resuspension in ~250 µl cold PBSE per gradient and layering on top of the pre-formed gradients. Gradients were centrifuged for 4 min at 2,000 g, 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 to pellet the cells, which were then re-suspended in 1ml PBSE per timepoint (re-uniting samples split across two gradients). Cells were briefly sonicated in a chilled Bioruptor 30 s on low power, then 25 µl Streptavidin microbeads added (Miltenyi Biotech 1010007) and cells incubated for 5 min on a wheel at room temperature. Meanwhile, 1 LS column per sample (Miltenyi Biotech 1050236) was equilibrated with cold PBSE in 4 °C room. Cells were loaded on columns and allowed to flow through under gravity, washed with 2 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. Cells were re-suspended in 50µl Lysis/Binding Buffer (from mirVANA kit, Life Technologies AM1560), and 50 µl 0.5µm 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, then 250µl Lysis buffer added followed by 15µl miRNA Homogenate Additive and cells were briefly vortexed before incubating for 10 minutes on ice. 300µl 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 13,000 g, 4 °C. Pellet was washed with cold 70% ethanol and re-suspended in 10 µl water. 500ng RNA was used to prepare libraries using the NEBNext Ultra Directional mRNAseq kit (NEB E7420, E7490, E7335, E7500, E7710) as described with modifications: Reagent volumes for RT, second strand synthesis, tailing and ligation were reduced by 50%, while libraries were amplified for 12 cycles using 2 µl each primer per reaction before two rounds of AMPure bead purification as described in the kit at 0.9x ratio prior to analysis by quality control using a Bioanalyzer.