Hiseq1000 system

The libraries were sequenced using the HiSeq1000 system (Illumina, San Diego, CA, USA). The generated sequences were analyzed using eXSP, an in-house pipeline designed to automate the analysis workflow, composed of modules performing every step using the appropriate tools available to the scientific community or developed in-house [33 (link)]. Paired sequencing reads were aligned to the reference genome (UCSC, hg19 build) using BWA and sorted with SAM tools and Picard (http://picard.sourceforge.net). Post alignment processing (local realignment around insertions-deletions and base recalibration) and SNV and small insertions-deletions (ins-del) calling were performed using the Genome Analysis Toolkit (GATK) [34 (link)] with parameters adapted to the haloplex-generated sequences. The called SNV and ins-del variants produced with both platforms were annotated using ANNOVAR [35 (link)] with; the relative position in genes using RefSeq [36 (link)] gene model, amino acid change, presence in dbSNP v137 [37 (link)], frequency in NHLBI Exome Variant Server (http://evs.gs.washington.edu/EVS) and the 1000 genomes large scale projects, multiple cross-species conservation and prediction scores of damaging on protein activity [38 (link)]. The annotated variants were then imported into the internal variation database.

The left nucleus accumbens from each rat was purified using the RNeasy kit using the manufacturer's directions. cDNA libraries were created by reverse transcribing the RNA and creating the second strand. Blunt ends were phosphorylated and “a-tailed” so that adapters could be ligated to both ends. RNA was sequenced with a HiSeq 1000 system from Illumina. Four samples were added to each lane. cDNA was amplified using “bridge” amplification. Base calls were made using fluorescently labeled nucleotides. Over 100 million reads were taken at 50 bp (paired-end reads). FastQC (v0.9.1) (Andrews, 2014 ) was used to check the quality of the reads. Reads were mapped to the rat reference genome (RN4) using Tophat2 (v2.0.4) (Kim et al., 2013 (link)) and Bowtie2 (v2.0.0.6) (Langmead and Salzberg, 2012 (link)) software packages. The R package EdgeR (v.3.0.8) (Anders and Huber, 2010 (link); Robinson et al., 2010 (link)) was then used for analysis using the “trimmed mean for M-values” (TMM) method for normalization and tag-wise dispersion using “count” data. A likelihood ratio F-test was used for generating P-values to compare EC vs. IC rats. Transcripts significantly regulated (p < 0.05) were overlaid onto the IPA energy metabolism pathway for comparison to protein values.

Per replicate and genotype 40 eye antennal discs or 30 wing discs from third instar larvae were dissected. RNA was extracted via peqGold MicroSpin Total RNA Kit. Library preparation and RNA-Seq were carried out according to the NEBNext Ultra RNA Library Prep protocol, the Illumina HiSeq 1000 System User Guide, and the KAPA Library Quantification Kit—Illumina/ABI Prism User Guide. Library preparation and RNA-Seq were performed at the Genomics Core Facility “KFB—Center of Excellence for Fluorescent Bioanalytics” (University of Regensburg, Regensburg, Germany).