GSM4066703: sml1_E_20h_rep1; Saccharomyces cerevisiae; RNA-Seq
Total mRNAs were extracted from 2x10^7 cells using the RNeasy mini kit (QIAGEN) according to the manufacturer's instructions. 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 µg of total RNA was poly-A selected using the Dynabeads mRNA Direct Micro Purification kit (Thermo Fisher Scientific, Cat# 61021) according to manufacturer's protocol. 50 ng of poly-A RNA were used to prepare strand-specific barcoded RNA libraries with the Ion Total RNA-Seq kit v2.0 (Thermo Fisher Scientific, Cat# 4475936). Poly-A RNA was fragmented with RNAse III, and purified with Nucleic Acid Binding Beads. RNA fragments were hybridized and ligated with Ion Adaptor and subsequently reverse transcribed into cDNA. The cDNA was amplified with the Ion Torrent barcode primer and purified with Nucleic Acid Binding Beads. Final libraries were quantified on the 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). Ten barcoded libraries were pooled together on an equimolar basis at a 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.
GEO Accession
GSM4066703