title: Old Mouse Flora Conventionalized #5 (young recipient)
design: Library preparation was performed according to Illuminas TruSeq Stranded mRNA protocol with the following modifications: The oligo-dT mRNA purification step was omitted and instead, 200ng of total RNA were directly added to the Elution2-Frag-Prime step. The PCR amplification step, which selectively enriches for library fragments that have adapters ligated on both ends, was performed according to the manufacturers recommendation but the number of amplification cycles was reduced to 12. Each library was uniquely tagged with one of Illuminas TruSeq LT RNA barcodes to enable library pooling for sequencing.The finished 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 in one lane on an Illumina HiSeq2500 sequencer in rapid mode at a read-length of 100bp paired-end.