title: Rat pineal gland, RNA-Seq, P40, ZT7
design: For the mRNA-Seq sample preparation, the NEB Master Mix Set was used according to the manufacturer's protocol. Briefly, 10 µg of each total RNA sample was used for polyA mRNA selection using streptavidin-coated magnetic beads, followed by Covaris 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 multiplex paired-end (PE) adaptors. Size selection was performed using a 1.5% cassette on a Pippin Prep (Sage Science). Finally, the libraries were enriched and barcodes incorporated using 14-16 cycles of PCR and purified Ampure XP bead (Agilent). The enriched libraries were diluted with Elution Buffer to a final concentration of 10 nM. Libraries were barcoded and four were run together in one lane at a concentration of 15 pM on an Illumina HiSeq 2000 using paired-end 101 base-pair sequencing, including a 7 cycle index read. Image analysis and base calling was done by Illumina Genome Analyzer Pipeline software (GAPipeline) with default parameters. All eight samples were analyzed in this manner.