GSM1201979: RNA-seq_spt6I; Schizosaccharomyces pombe; RNA-Seq
Total RNA was isolated from S. pombe cells using a hot phenol method followed by phenol-chloroform extractions, precipitation, and purification using Qiagen RNeasy columns. 10 μg of total RNA were used as starting material for strand-specific library preparation using the Illumina Universal RNA-seq samples prep kit.  This kit is an unreleased early version provided by Illumina of the Illumina TruSeq Small RNA Sample Prep Kit.  Briefly, poly(A)+ RNA was enriched by two rounds of poly(dT) Sera-Mag magnetic beads purification, then fragmented to an average size of ~200 nucleotides.  Fragmented RNA was 3’ de-phosphorylated with Antarctic phosphatase and 5’ phosphorylated with polynucleotide kinase to prepare RNA fragments for subsequent ligation.  Illumina RNA adaptors were ligated to the 5’ and 3’ ends using a 3’ RNA ligase and a T4 RNA ligase, respectively.  First strand cDNA was produced using a primer specific for the Illumina 3’ adaptor.  The library was amplified with 15 cycles of PCR using primers specific for the Illumina adaptors and purified using SPRI-beads (Agencourt, Beckman Coulter).  Library size distributions and concentrations were determined on an Agilent Bioanalyzer.  RNA-seq libraries were sequenced on an Illumina Genome Analyzer IIx instrument.