alias: E-MTAB-5836:JCSEQ49_s
title: AB SOLiD 4 System sequencing; Effect of stress on the binding affinity of yeast eIF4F components to their target RNAs
design: Effect of stress on the binding affinity of yeast eIF4F components to their target RNAs
library_construction_protocol: Strains were grown at 30°C in synthetic complete dextrose media lacking histidine [SCD–his] grown to A600=~0.6.  Addition of 0.4 mM H2O2 (+H2O2 for 15 min) prior to cell harvest. Yeast cultures were grown to A600=~0.6, pelleted, snap frozen in liquid nitrogen and ground in Buffer A (20 mM Tris-HCl [pH 8], 140 mM NaCl, 1 mM MgCl2, 0.5% NP40, 0.5 mM DTT, 1 mM PMSF, EDTA free Protease Inhibitor cocktail tablet (Roche), 100 µM NaV3O4, 5 mM NaF and 40 units/ml RNAsin) using liquid nitrogen and a 6870 Freezer Mill (Spex). Lysates were cleared through two centrifugation steps (15,000 x g at 4°C) and quantified using Bradford Reagent. 10 mg total protein was loaded onto Rabbit IgG coupled Tosyl-activated Dynabeads M-280 magnetic beads (Dynal) to ensure maximum depletion of the tagged protein from each extract. After the final wash, the beads were re-suspended in 270 µl Buffer A. A 20 µl aliquot of the sample was set aside for western blot analysis, and RNA was purified from the remaining 250 µl for RNAseq.   Triplicate samples of TAP associated RNA was isolated on M-280 magnetic beads as described above. For RNA target identification, after the final wash, the beads were resuspended in 270 µl Buffer A. A 20 μl aliquot of the sample was set aside for western blot analysis, and RNA was purified from the remaining 250 μl. Total (T) RNA and paired immune precipitated (IP) RNA were isolated using a standard Trizol Reagent (Life Technologies, Carlsbad, CA) protocol and re-suspended in 10 µl diethylpyrocarbonate (DEPC) treated water. RNA was quantified using a Nanodrop 8000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). rRNA was then depleted from both total and IP RNA samples using the RibominusTM Eukaryote Kit for RNA-Seq (Life Technologies, Carlsbad, CA). Depleted samples were ethanol precipitated, washed twice with 70% ethanol and resuspended in 10 µl DEPC water. rRNA depletion was assessed on a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) using a RNA nanochip and the remaining RNA stored at –80°C. Sequencing libraries were generated using the whole Transcriptome Library Preparation protocol provided with the SOLiD® Total RNA-Seq Kit (Life Technologies, Carlsbad, CA) as described by the manufacturer.
identifiers.namespace: Division of Cardiovascular Sciences School of Medicine Faculty of Biology Medicine and Health Manchester Academic Health Science Centre The University of Manchester
identifiers.id: E-MTAB-5836:JCSEQ49_s