GSM4182903: D1/8 KLY194 Rep1_RPF_Run2; Saccharomyces cerevisiae; RNA-Seq
750ml of yeast cultures were grown to OD600 0.6-0.7 un/ml at 25°C. Cycloheximide was added to final concentration of 0.1 mg/ml, and the cultures were put back to incubate for the additional two minutes. Treated cells were harvested by centrifugation at 4°C. Pellets were homogenized in the ice-cold lysis buffer (1X Yeast Polysome Buffer, 1% Triton X-100, 0.1 mg/ml Cycloheximide). For that, 1.5 ml of lysis buffer and 5 g of glass beads were added for each gram of pellet. Cells were lysed by vortexing 6x 20 seconds and incubated on ice between cycles. Crude extract was spun at 3,000 x g for 5 min, the supernatant was collected to chilled 1.5 ml tubes on ice, and the lysate was clarified by centrifugation for 10 minutes at 20,000 x g in a 4°C centrifuge. The supernatant was recovered avoiding both the pellet and the lipid layer at the top of the tube. DNase UI was added to a final concentration of 10 U/ml. For each lysis, A260 was adjusted to 200, and the 100 µl aliquot was kept for the Total RNA extraction and 200µl aliquot was used for the Ribosome Protected Fragment RNA preparation. 10µl of 10% SDS was added to the 100µl aliquot, and the sample was snap frozen in liquid nitrogen and kept at -80oC until ready for use. 0.3 units of Truseq Nuclease per A260 of lysate was added to the 200µl aliquot of clarified lysate. Reaction was incubated at room temperature for 60 minutes with gentle mixing. Nuclease digestion was stopped with 10 µl of SUPERaseIn RNase Inhibitor (Life Technologies, cat. No. AM2696) and chilled on ice. Truseq™ Ribosome Profiling kit (Illumina, formerly ArtSeq) was used for obtaining ribosome-protected RNA fragments using size-exclusion chromatography columns.  Both RPF and total mRNA input fragments were cloned according to the manufacturer protocol.
GEO Accession
GSM4182903