AB 5500xl Genetic Analyzer sequencing; Analysis of the effect of PUF3 deletion in mRNA translation in yeast
Analysis of the effect of PUF3 deletion in mRNA translation in yeast
Cycloheximide was added to a final concentration of 0.1 mg/ml, and yeast were harvested by centrifugation. Saccharomyces cerevisiae were lysed into polyribosomal buffer containing cyclohexamide and 9 OD260 units were loaded onto a sucrose gradient.  15-50% sucrose gradients were poured.  15 fractions were collected from each gradient.  Fractions 4-8 were pooled to form the monosome sample and 10-15 were pooled to form the polysome sample. Grown in SCD-His media at 30oC to exponential phase (OD600 0.5-0.7). Samples lysed in Buffer A using a 6870 Freezer Mill (Spex). Lysates cleared by centrifuging twice at 15000 g. Nucleic acids extracted using Trizol Reagent. rRNA was depleted from samples using RibominusTM Eukaryote Kit for RNA-Seq (Invitrogen). Depleted samples were precipitated with 2.5x volumes ethanol, 1/10th volume 3M sodium acetate and 1μl glycogen, washed twice with 70% ethanol and re-suspended in 10 μl DEPC water.  Sequencing libraries were generated using the whole Transcriptome Library preparation protocol provided with the SOLiD® Total RNA-Seq Kit.