Cells were grown overnight in a 30 C shaker in 500 ml of YPAD media to mid-log phase (OD 595 0.8-1.5), cooled to 4 C for 1 h to allow ribosomes to run off of transcripts while remaining tightly coupled. Cells were harvested by centrifugation and washed three times with 40 ml 0.9% KCl solution. Cell pellets were stored at À80 C until needed, at which time they were thawed and resuspended in 1 ml binding buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 60 mM NH 4 Cl, 2 mM DTT, 1 mM PMSF) per gram of cells. Cells were lysed with a 1:1 vol of Zirconian beads (BioSpec, Bartlesville, OK, USA) and disrupted using two 2-min pulses of a minibead beater. Lysates were clarified by centrifugation at 20 000 r.p.m. (50 000 g) using an MSL-50 rotor at 4 C for 25 min. Ribosomes were chromatographically purified using Sulfolink beads (Pierce, Rockford, IL, USA) as previously described (26) , and eluted from the resin in 8 ml of elution buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 500 mM KCl, 2 mM DTT, 0.5 mg/ml heparin). Eluted ribosomes were treated with 2 mM puromycin and 1 mM GTP for 30 min at 30 C and were layered on top of a 22-ml glycerol cushion [50 mM HEPES-KOH pH 7.6, 10 mM Mg(CH 3 COO) 2 , 50mM NH 4 Cl, 1 mM DTT, 25% glycerol] and pelleted by centrifugation at 30 000 r.p.m. at 4 C for 18-20 h. Pellets were washed with 1 ml of storage buffer [50 mM HEPES-KOH pH 7.6, 10 mM Mg(CH 3 COO) 2 , 50mM NH 4 Cl, 1 mM DTT, 25% glycerol], and resuspended in 200-400 ml of storage buffer. Concentrations were determined spectrophotometrically (1 OD 260 = 20 pmol ribosomes). The salt-washed ribosomes were aliquoted and stored at À80 C for up to 3 months. Ribosomal rRNA quality was checked on 1.3% agarose gels and rRNA to protein ratios were monitored by determining OD 260 to OD 280 ratios. Polysome profiles were obtained by sucrose density gradient centrifugation as previously described (27) . Samples were split, and 10 ml of dimethyl sulfoxide (DMSO) was added to half of the samples, while 10 ml of 60 mM 1M7 was added to the other half. Samples were incubated at 30 C for 20 min. Ribosomes were precipitated by the addition of 275 ml of ice-cold 100% ethanol and stored at À20 C for 1-2 h. Ribosomes were pelleted by centrifugation at 10 000 r.p.m. for 2 min and resuspended in lysis buffer and rRNAs were isolated using an Ambion (Austin, TX, USA) RNAqueous Õ -Micro RNA isolation kit. Optical densities were taken at 260 nm and 280 nm to monitor the quantity and quality of RNA, and samples were resuspended at a concentration of 1 mg rRNA/7 ml in pure water. HPLC purified oligonucleotide primers purchased from IDT (Coralville, IA, USA) are listed in Supplementary Table 2. Oligonucleotides were resuspended to 25 pmol/ml, 3 0 end labeled with g[ 32 P]ATP with T4 polynucleotide kinase (Roche, Indianapolis, IN, USA), and purified from free radiolabeled nucleotide by passage through a MicroSpin G-25 column (GE Healthcare, Piscataway, NJ, USA). Annealing reactions utilized 1 mg of modified rRNAs and 3 ml of labeled oligonucleotide heated at 80 C for 1 min, followed by a 4-7-min incubation at 5-10 C below the T m of each oligonucleotide. Annealed rRNA/ primers (2 ml each) were added to 3 ml of cold enzyme mix [0.25 ml 10 mM dNTP, 0.25 ml 100 mM DTT, 1 ml 5X Superscript III Buffer, 0.25 ml Superscript III (Invitrogen Life Technologies, Carlsbad, CA, USA), 1.25 ml H 2 O]. For sequencing samples, an additional 1 ml of each ddNTP was added to each C, T, A, G, sample, respectively. Primer extension reactions were performed at 52 C for 25 min, with potential 2-min-long extensions preceding the 52 C at lower temperatures depending on the individual T m values of the primers. Denaturing RNA loading dye (2 ml) was added to each sample, heated to 94 C for 2.5 min, and samples were resolved through 6% urea-acrylamide denaturing gels. Gels were dried and radiolabeled samples were visualized by phosphorimagery.