AB SOLiD 4 System paired end sequencing; Analysis of the role of yeast Puf3p in translation control
Analysis of the role of yeast Puf3p in translation control
Grown in SCD-His media at 30oC to exponential phase (OD600 0.5-0.7), pelleted by centrifugation and snap frozen into liquid nitrogen. 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. The aqueous phase was mixed with 70% ethanol and the RNA was purified using RNeasy Minikit (Qiagen). Beads were pre-washed thrice with Buffer A and then added to 4mg/ml of lysate. Puf3p-TAGged samples were immunoprecipitated for 20 minutes at 4oC and washed five times with Buffer A containing 10 units/ml RNAsin, changing tubes twice during the washes and the final two washes were performed for 15 minutes each.  After the final wash the beads were resuspended in 250 µl Buffer A and treated with Trizol.  The aqueous phase was mixed with 70% ethanol and the RNA was purified using RNeasy Minikit (Qiagen). 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