GSM7506498: Yeast Polysome Profiling R2, Fraction 20; Saccharomyces cerevisiae; RNA-Seq
To start the cell lysis, cells were dissolved in 1X Polysome Lysis Buffer (PLB) [20mM Tris-HCl (ThermoScientific #AM9856), 10 mM MgCl2 (ThermoScientific #AM9530G), 1mM DTT (ThermoScientific #707265ML), 10 µg/mL cycloheximide (ThermoScientific #J66004-XF), 2 cOmplete EDTA free Protease Inhibitor (Sigma #COEDTAF-RO)/100mL, 8U/ml RNasIN (Promega #N2611), 1% v/v Triton X-100 (Fluka #93418)]. Lysis was carried out with Zirconia/Silica beads (Biospec #11079105z) with cell suspensions loaded in 1.5mL microfuge tubes on benchtop shaker/incubator set at 4°C. 15 cycles of interval shaking was carried out with each cycle consisting of 30s ON at 2000rpm and 30s OFF. For the gradient, 10% and 50% w/v sucrose (ThermoScientific #036508-30) solutions were prepared in 1x Polysome Gradient Buffer (PGB) [as PLB but without Triton X-100]. The 10% sucrose was loaded on the centrifuge tubes (Beckman C14293/344059 Ultraclear 13.2ml [compatible with SW41Ti rotor]). With a 20ml syringe attached to long cannula, the 50% solution was layered at the bottom. The tubes were then covered with caps (BioComp #105-414-1) and the gradient was formed by high angle rotation, using the 10%-50% short cap (rate zonal) protocol in the BioComp™ Gradient Station machine. To assess the volume of the cell lysate to be loaded, A254 was measured and a total of 10-50 A260 units was loaded onto the 10-50% sucrose gradient. Ultracentrifugation was carried out with a SW41Ti rotor (swing bucket) at 40000rpm for 2h at 4°C. The polysome profile was generated using the BioComp™ Gradient Station machine and 25 different fractions (450µL each) corresponding to different lengths in the centrifuge tube were collected. RNA from each fraction was purified by acid Phenol : Chloroform : Isoamyl Alcohol :: 25 : 24 : 1. Precipitation carried out with one volume of isopropanol at 4°C for 30 min to 1h with 2µL of GlycoBlue (ThermoScientific #AM9516) as co-precipitant. RNA was reconstituted with 20µL of nuclease free water (ThermoScientific #AM9932) containing RNasIN (Promega #N2611) at 1µL/mL (40U/mL). Stranded SMART-Seq (Takara)