title: Transcriptome from 2dpf keratocytes, replicate 3
design: RNA was extracted from approximately 1 x 105 keratocytes per sample using Trizol (Invitrogen).  Three biological replicates were collected for each developmental stage.  Ribosomal RNA was depleted using the Ribo-Zero magnetic kit (Epicentre).  The remaining mRNA was then fragmented with 8 minutes incubation in 50mM sodium carbonate/bicarbonate, 1mM EDTA, pH 9.2 at 95 degrees.  To prepare cDNA, first strand synthesis was performed with Superscript III (Invitrogen) using random hexamer priming, and second strand synthesis was performed with DNA Polymerase I (NEB).  Illumina libraries were prepared from the cDNA in an automated fashion using the Illumina TruSeq sample prep kit with TruSeq adapters on a SPRIworks System I (Beckman Coulter).  Sequencing reactions were performed on an Illumina Genome Analyzer IIX according to manufacturer’s instructions to generate 40nt single-ended reads.