title: In vitro rat Pituitary RNA-sequencing project.
design: For the mRNA-Seq sample preparation, the Illumina standard kit was used according to the manufacturer's protocol. Briefly, 1 µg of each total RNA sample was used for polyA mRNA selection using streptavidin-coated magnetic beads, followed by chemical mRNA fragmentation. The fragmented mRNA was used to synthesize cDNA  using reverse transcriptase (SuperScript II) and random primers. The cDNA was further converted into double stranded cDNA and, after an end repair process (Klenow fragment, T4 polynucleotide kinase and T4 polymerase), was finally ligated to Illumina paired-end (PE) adaptors. Size selection was performed using a 2% agarose gel. Finally, the libraries were enriched using 15 cycles of PCR and purified by the QIAquick PCR purification kit (Qiagen). The enriched libraries were diluted with Elution Buffer to a final concentration of 10 nM. Libraries were barcoded and four were run at a concentration of 7 pM on one Illumina HiSeq 2000 lane using paired-end 101 base-pair sequencing. Image analysis and base calling was done by Illumina Genome Analyzer Pipeline software (GAPipeline) with default parameters.