title: RNA-seq for antP mutant biological replicate 1
design: Double-stranded cDNA was fed into Illuminas TruSeq Stranded mRNA protocol. Twelve-cycles of PCR amplification was performed to enrich the library fragments with adapters on both ends. Each library was uniquely tagged with one of Illuminas TruSeq LT RNA barcodes to enable library pooling for sequencing. Libraries were quantitated using Invitrogens Picogreen assay and the average library size was determined on a Bioanalyzer 2100, using a DNA 7500 chip (Agilent). Library concentrations were then normalized to 4nM and validated by qPCR on a ViiA-7 real-time thermocycler (Applied Biosystems), using qPCR primers recommended in Illuminas qPCR protocol, and Illuminas PhiX control library as standard. The libraries were then pooled at equimolar concentrations and sequenced on an Illumina HiSeq2500 sequencer in rapid mode at a read-length of 100 bp paired-end reads.