alias: E-MTAB-2567:SLF1-IP-3
title: AB SOLiD 4 System sequencing; Analysis of the role of yeast Slf1p in translation of specific mRNAs during oxidative stress response
design: Analysis of the role of yeast Slf1p in translation of specific mRNAs during oxidative stress response
library_construction_protocol: Grown in SCD media at 30C to exponential phase (OD600 0.5 - 0.7), pelleted by centrifugation and snap frozen into liquid nitrogen Samples lysed in Lola lysis buffer using a 6870 Freezer Mill (Spex). Lysates cleared by centrifugating twice at 15000 g. Nucleic acids extracted using Trizol Reagent. Slf1p-TAGged samples were immunoprecipitated for 20 minutes at 4 C and washed five times with Lola buffer containing 10 units/ml RNAsin, changing tubes at least twice during the washes and the final two washes were performed for 15 minutes each.  For RNA isolation after the final wash, the beads were resuspended in 250 ul Lola Buffer and treated with Trizol. The aqueous phase was mixed with 70% ethanol and the RNA was purified using the RNeasy minikit (Qiagen). rRNA was depleted from samples using Ribominus Eukaryote Kit. Depleted samples were precipitated with 2.5x volumes ethanol, 1/10th volume 3M sodium acetate and 1_l glycogen, washed twice with 70% ethanol and re-suspended in 10_l DEPC water.  Sequencing libraries were then generated using the whole Transcriptome Library Preparation protocol provided with the SOLiD Total RNA-Seq Kit. cDNA was then amplified and barcoded with SOLiD RNA barcoding Kit and samples purified using PureLink PCR Micro Kit
identifiers.namespace: THE UNIVERSITY OF MANCHESTER
identifiers.id: E-MTAB-2567:SLF1-IP-3
identifiers.namespace: The University of Manchester
identifiers.id: E-MTAB-2567:SLF1-IP-3