title: Salaria pavo transcriptome assembly and gene expression
design: Total RNA was extracted from whole brain tissue for each individual of the four phenotypes. Samples were transferred to 1 ml of QIAzol lysis reagent (Qiagen) and the tissue homogenized (on ice) with a sonicator (Sonopuls HD 2070; Bandelin, Berlin, Germany) during 30 s at 20-30% power. RNA was then extracted using the RNeasy Lipid Tissue Mini Kit (Qiagen) following manufacturer’s protocol. RNA quality and concentration for each sample were determined using a NanoDrop 1000 spectrophotometer (Thermo Scientific). Equal amounts of RNA from each individual were used to prepare the RNA pools corresponding to the high and low GSI libraries for each phenotype.  For expression analysis, preparation and sequencing of the eight pooled RNA libraries was performed by The Genome Analysis Centre (Norwich, United Kingdom). Upon arrival, samples’ RNA integrity was first verified on Agilent 2100 Bioanalyzer. Libraries were prepared from polyA+ RNA and sequenced in two lanes on the Illumina HiSeq 2000 as paired-end 100 bp reads with an approximate insert size of 341 bp. Each library was sequenced at a high depth in order to detect transcripts that can become under-represented when analysing whole brain tissue expression. To improve the de novo assembly, four additional pooled libraries, one for each phenotype, were also prepared using RNA from the previous collection, and sequenced using Roche’s 454 technology. Library preparation and single-end sequencing on a GS-FLX System were performed at Max Planck Institute (Berlin, Germany) using the manufacturer's protocol.