title: Whole transcriptome profiling of the rat pineal gland at mid-night ZT19 (Experiment 3)
design: A SPRI-TE kit (Beckman-Coulter) was used wherein 10 µg of total RNA was polyA-RNA-enriched using streptavidin-coated magnetic beads, followed by Covaris RNA fragmentation. Fragmented RNA was used to generate 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 ligated to Illumina paired end (PE) adaptors containing a 6-basepair bar-code. Size selection was performed using a Pippin Prep 1.5% cartridge. Libraries were enriched using 16 cycles of PCR and purified using Ampure XP Beads (Agencourt). Each library was run as one of six bar-coded samples per lane on an Illumina HiSeq machine yielding paired-end,  101-base sequencing reads. Image analysis and base calling were done using RTA1.12.4.2 and CASAVA1.8.0.