alias: GSM1919543
title: GSM1919543: sk1-1; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: RNA isolation for RNA-seq was performed as follows. RNA was isolated similar to Clarkson, B. K., Gilbert, W. V. & Doudna, J. A. Functional overlap between eIF4G isoforms in Saccharomyces cerevisiae. PLoS ONE 5, e9114 (2010). Pellets were resuspended in 1mL Acid Phenol and an equal volume of AES buffer (50mM NaAcetate pH 5.2, 10mM EDTA, 1% SDS) was added. In 2mL eppendorf tubes, samples were incubated at 65°C for 10 min with vortexing every minute. Samples were incubated on ice for 5 min and then transferred to a phaselock tube and one volume Cholrofom was added. After spinning, the top aqueous layer was transferred to a fresh phaselock tube and one volume of phenol:choloform:iaa (25:24:1) was added, tubes were spun, one volume of choloform was added, tubes were spun, and the aqueous layer was transferred to a fresh tube to be precipitated with 50uL 3M NaOAc (pH 5.5) and 550uL isopropenol. Samples were spun at max speed for 25 minutes at 4°C. The pellet was washed twice with 70% ethanol and resuspended in water. RNA was isolated using the hot acid phenol method and DNase treated with Turbo DNase. rRNA was subtracted using the Illumina Ribo-Zero Gold rRNA Removal Kit (Yeast). RNA was then fragmented and first strand cDNA was performed. Second strand synthesis was carried out using dUTP instead of dTTP to generate strand specific libraries. Subsequently adaptors ligation, uracil digestion, and PCR were performed. All libraries were barcoded and sequenced in one NextSeq lane. RNA-seq (Ribo-Zero, strand specific)
identifiers.namespace: GEO
identifiers.id: GSM1919543