GSM8566361: Total ribosomes profiling (Reh1), rep1; Saccharomyces cerevisiae; OTHER
Cells were washed once in IP buffer (Tris HCl 20 mM pH 7.5, 100 mM KCl, 10 mM MgCl2, 100 ug/ml CHX, Pierce Protease Inhibitor Cocktail, EDTA-free (1 tablet/10 ml), 1 mM PMSF, 1 uM each leupeptin and pepstatin and 5 mM beta-mercaptoethanol (BME)) and then resuspended in a volume of IP buffer equal to that of the cell pellet. Extracts were prepared by agitation with glass beads, Igepal was added to 0.1% extracts were clarified by centrifugation at 4°C for 15 minutes at 18,000 g. 20 mg of RNA (~2 ml sample) was digested with 62.5 units of RNase I (Epicentre) at 4°C for 1 hr. 50ul was removed for total Ribo-seq sample. 25 ul bed volume washed anti-FLAG magnetic beads (Sigma Milipore) was added to the remaining sample and samples were rotated for 1 hr at 4°C. Beads were washed 4 times with IP buffer supplemented with 0.1% Igepal and transferred to a clean tube during the last wash. Reh1-bound ribosomes were eluted in 100 ul of IP buffer supplemented with 0.1% Igepal and 150 ug/ml 3xFLAG peptide (Sigma Milipore) for 15 min at 4°C. 12.5 units RNase I was added to the eluate and digestion was carried out at room temperature for 45 min. RNAse I digestion reaction was stopped by addition of QIAzol followed by RNA extraction by chloroform and ethanol precipitation. For total RIbo-seq samples, 6.25 units RNase I (Epicentre) was added to 5ul of sample in a total volume of 50 ul in IP buffer supplemented with 0.1% Igepal. Digestion was carried out at room temperature for 45 min. Digested lysates were layered onto 50 ul 1M sucrose cushions prepared in IP buffer and ribosomes were pelleted by centrifugation at 70,000rpm in a TLA 100 rotor (Beckman Coulter) for 30 min at 4°C. RNA was extracted from the ribosome pellets with the addition of 700 μl QIAzol followed by chloroform and ethanol precipitation. Isolated RNAs were size-selected using a 15% polyacrylamide TBE-UREA gel (Invitrogen) and 28-35 nt fragments were excised and extracted by crushing the gel fragment in 400 µL of RNA extraction buffer (300 mM NaOAc [pH 5.5], 5 mM MgCl2). The sample was passed through a Spin X filter (Corning 8160) and the flow through was ethanol precipitated (2.5 X volume) with 1.5 µL of Glycoblue (Invitrogen AM9516). Ribosome profiling libraries were prepared as previously described43 using the D-Plex Small RNA-Seq Kit (Diagenode). The cDNA was amplified for ten cycles. Each library was cleaned with PCR purification kit (Qiagen) and eluted with 30 μL of RNase-free water. To completely remove the primer-dimers, size-selection was done in a 3% agarose pre- cast gel (Sage Science) with the BluePippin system (Sage Science) using 180-235 nt range (tight settings, target:208 bp). The resulting size-selected libraries were analyzed with the Agilent High Sensitivity DNA Kit (Agilent). Each library was mixed in equimolar proportion for sequencing with the NovaSeq 6000 SP PE.