title: GSM8792348: Yeast, PolysomeProf, R1, 42C, NonDepl, Pool3; Saccharomyces cerevisiae BY4743; RNA-Seq
library_name: GSM8792348
library_construction_protocol: Cells were collected by addition of 1 volume of culture to 2 volumes of chilled medium (0°C) containing cycloheximide (CHX) (ThermoScientific #357420050) at 1.5x (150 µg/mL). The resulting mixture was immediately submerged in a -20°C bath (1:1 mix of ethanol and water) for two minutes (so that the final temperature is less than 10°C) The cells were spun down at 4000rpm (3000g) for 10min in a centrifuge at 4°C. The supernatant was removed and the pellet was immediately subjected to lysis. The cell lysis and gradient preparation was performed as previously described in Rahaman, et. al. Science Advances, 2023. For the ultracentrifugation, the A254 of the lysate was measured by mixing sample (or blank with Polysome Lysis Buffer) with water in a 1:20 volumetric ratio. 2-4 A254 unit equivalents of  the sample was loaded onto the 10-50% sucrose gradient. Ultracentrifugation was carried out with a SW41Ti rotor (swing bucket) at 40000rpm (equivalent to 275,000g) for 2h at 4°C. The polysome profile was generated with BioComp™ Polysome Profiling machine. 25 fractions (450µL each) corresponding to different positions in the centrifuge tube were collected. For RNA purification, the fractions were pooled according to the peaks of A254 polysome profile (free fraction, 40S, 60S, monosome, etc. and in-vitro transcribed spike-in RNAs were added to each pool to serve as a volumetric control. Subsequently, the pools were subjected to phenol-chloroform extraction (Phenol : chloroform : isoamyl-alcohol = 25:24:1). Precipitation was carried out with 2.5 volumes of ethanol at −80°C overnight 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). This RNA was further purified using RNeasy MinElute Cleanup Kit (Qiagen #74204) with on-column DNase digestion. The purified RNA was then used for RNA-Seq. Stranded SMART-Seq (Takara)