title: GSM3141809: Top2-1-37degree; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: RNA was isolated from 5X10e7 cells by using QIAGEN Rneasy mini kit (50) (Cat no 74104) QIAGEN. Prior to library preparation cytoplasmic and mitochondrial ribosomal RNA was removed by using "Ribo-Zero Gold rRNA Removal Kit (Yeast) cat no. MRZY1324" Libraries for RNA sequencing  were prepared following the manufacturer protocols for transcriptome sequencing with the Ion Proton sequencer (Thermo Fisher Scientific/Life Technologies). Briefly 1 ug of Total RNA was poly-A selected using the Dynabeads mRNA Direct Micro Purification kit (ThermoFisher, catalog no. 61021) according to manufacturer's protocol. About 50 ng of poly-A RNA were used to prepare strand-specific barcoded RNA libraries with the Ion Total RNA-Seq kit v2.0 (ThermoFisher Scientific, catalog no. 4475936). Briefly poly-A RNA were fragmented with RNAse III and purify with Nucleic Acid Binding Beads.  After purification the poly-A RNA fragmented were hybridized and ligated with Ion Adaptor and subsequently reverse transcribed for cDNA preparation.  cDNA were amplified with Ion Torrent barcode primer and purified with  Nucleic Acid Binding Beads.Final libraries were quantified on Qubit fluorometer with HS DNA (Thermo Fisher Scientific/Life Technologies) and checked for size on an Agilent Bioanalyzer with HS DNA kit (Agilent, Santa Clara, CA). Four barcoded libraries were pooled together on an equimolar basis according to the final concentration of 11 pM and clonally amplified using the Ion Proton™ Hi Q™Template Kit (Thermo Fisher Scientific/Life Technologies) with IonOneTouch 2 instrument (Thermo Fisher Scientific/Life Technologies) . After emulsion PCR, DNA positive ISPs were recovered and enriched according to standard protocols with the IonOneTouch ES Instrument (Thermo Fisher Scientific/Life Technologies). A sequencing primer was annealed to DNA positive ISPs and the sequencing polymerase bound, prior to loading of ISPs into Ion P1 sequencing chips. Sequencing of the samples was conducted according to the Ion Proton™  Hi-Q  Sequencing Kit Protocol on Ion Proton instrument.
attributes.tag: GEO Accession
attributes.value: GSM3141809