GSM9306640: Total ribosome profiling (uL16 loop mutant), rep2; Saccharomyces cerevisiae; OTHER
For cell lysis, frozen pellets were mixed with frozen IP buffer (20 mM Tris·HCl, pH 7.5, 100 mM KCl, 10 mM MgCl2, 100 μg/mL cycloheximide,100 μg/mL Tigecyclin (Sigma), Pierce Protease Inhibitor Cocktail, EDTA-free [1 tablet/10 mL], 1 mM PMSF, 1 μM each of leupeptin and pepstatin, and 5 mM β-mercaptoethanol) at approximately 1 mL buffer per pellet. Samples were homogenized in a cryogenic mill (Retsch MM 300) for six cycles of 3 min each at 15 Hz, with samples submerged in liquid nitrogen for 3 min between cycles. The resulting cell powder was thawed on ice, additional IP buffer was added, and the lysate was clarified by centrifugation at 18,000 × g for 20 min at 4 °C. The clarified extract was supplemented with 0.1% (v/v) Igepal CA-630. The lysates were diluted to 150 A260 units/mL. One milliliter of lysate was treated with RNase I (Ambion) at a ratio of 3 U RNase I per 1 A260 unit for 1 h at 4 °C. A 50 μL aliquot of this extract was reserved as the total ribosome profiling sample. The remaining lysate was diluted to 50 A260 units/mL and incubated with 20 μL of pre-washed anti-FLAG magnetic beads (Sigma Millipore) per 1 mL of lysate for 2 h at 4 °C with rotation. Beads were washed three times with IP buffer containing 0.1% Igepal and transferred to a clean tube during the final wash. Bound ribosomes were eluted in 100 μL of IP buffer supplemented with 0.1% Igepal and 150 μg/mL 3×FLAG peptide (Sigma Millipore) for 30 min at 4 °C. Eluted ribosomes were digested with RNase I (1 U/μg RNA; Ambion) for 45 min at room temperature. Reactions were stopped by addition of QIAzol (Qiagen), and RNA was extracted using chloroform extraction followed by ethanol precipitation. Total ribosome profiling samples were processed in parallel by treating 50 μL of lysate with RNase I (1 U/μg RNA) in IP buffer supplemented with 0.1% Igepal for 45 min at room temperature. Digested lysates were layered onto 50 μL of 1 M sucrose cushions prepared in IP buffer, and ribosomes were pelleted by centrifugation at 165,000 × g for 30 min at 4 °C in a TLA 100 rotor (Beckman Coulter). RNA was extracted from ribosome pellets using 700 μL of QIAzol, followed by chloroform extraction and ethanol precipitation. To isolate the ribosome-protected fragments (RPFs), the digested RNA   was run on a 15% TBE–urea polyacrylamide gel (Invitrogen).  A 19–35 nt fragments were excised and extracted by crushing the excised gel slice in 400 μL RNA extraction buffer (300 mM sodium acetate, pH 5.5, 5 mM MgCl2 followed by incubation at -20 C for 1h and overnight incubation at RT on a hula mixer.  The gel extract was passed through a Spin-X filter (Corning 8160), and RNA was recovered from the flow-through by ethanol precipitation (2.5X volume) with 1.5 μL GlycoBlue (Invitrogen AM9516). Ribosome profiling libraries were prepared as previously described60 using the D-Plex Small RNA-Seq Kit (Diagenode). cDNA was subjected to rRNA depletion, and the cleaned-up cDNA was amplified by PCR for 12 cycles. Libraries were purified with AMPure XP (Beckman Coulter) at a ratio of 1:1.8 (sample to bead ratio) and eluted in 50 μL of water. The library quality was assessed using a Bioanalyzer and the Agilent High Sensitivity DNA Kit (Agilent). Final libraries were pooled in equimolar ratios and sequenced on the NovaSeqX Plus; PE 150 (Illumina).