GSM1368288: WT_180_r1; Schizosaccharomyces pombe; RNA-Seq
Total RNA was extracted from the fission yeast cells as follows: cells were treated with hot acidic phenol-chloroform, followed by phenol-chloroform extraction and RNA precipitation. Then RNA was column purified using QIAGEN RNeasy Kit 74104 according to the manufacturers instructions. RNA quality was determined using an Agilent 2100 Bioanalyser. Ribosomal reduction of total RNA (8μg) was performed using the Ribominus Eukaryote Kit for RNA-Seq (Invitrogen A10837-08) and the Ribominus Concentration Module (Invitrogen K155005). Greater than 90% ribosomal reduction was achieved for all samples when assessed on the Agilent 2100 Bioanalyzer. Libraries were prepared with 350ng of ‘ribosomally-reduced’ total RNA using the SOLiD Total RNA-Seq Kit (Life Technologies 4445374). We used the chemical hydrolysis method to fragment the RNA and performed size selection on Novex 6% TBE-Urea Gels (Invitrogen EC6865). Samples were multiplexed by barcoding with the SOLiD Transcriptome Multiplexing Kit (Life Technologies 4427046). Library quantitation was carried out with the Quant-IT DSDNA HS Assay Kit (Invitrogen Q32851) on the Qubit Fluorometer (Invitrogen Q3287). Molarities were determined using a High Sensitivity DNA Assay (Agilent Technologies 5067-1504) before pooling the libraries in equimolar quantities. Emulsion PCR was carried out on the pooled libraries using an input of 0.5pM in the EZ Bead System to prepare template beads following the manufacturer’s instructions  (Life Technologies PN 4448417). Templated beads were then sequenced on a 5500XL Genetic Analyser (Life Technologies 4460730) according to manufacturer’s instructions to generate 50 bases strand-specific reads.
GEO Accession
GSM1368288