title: Exploring the gonad transcriptome of two extreme male pigs with RNA-seq
design: Total RNA from gonads was extracted as described in [2]. Briefly, Total RNA was extracted from 100 mg tissue using the RiboPure™ kit (Ambion, Austin, USA). RNA integrity was assessed by Agilent Bioanalyser 2100 and RNA Nano 6000 Labchip kit (Agilent Technologies, Palo Alto, USA). Due to high variation in concentrations of the total RNA obtained in different tissues, all samples were concentrated and cleaned using the RNAeasy MiniElute Cleanup kit (Qiagen, Basel, Switzerland) obtaining final concentrations between 500 and 1000 ng/µl. Sequencing libraries were produced using the Illumina mRNA-Seq sample preparation kit, following the manufacturer’s instructions. Briefly, 4 ?g of total RNA were used as input for poly-A+ selection, followed by metal-catalyzed fragmentation of the selected mRNA (peak of size distribution at approx. 240 nt). After reverse transcription to cDNA using random hexamer primers, we performed end-repair and A-tailing of the double stranded cDNA. Large White and Iberian cDNA were ligated to indexed pairs of adapters, see Additional file 7. The cDNA was size selected on a 2% agarose gel, and fragments corresponding to an insert size of 237 nucleotides were excised from the gel. The DNA was recovered from the gel slice using QIAquick gel extraction kit (Qiagen). Therafter, the libraries were amplified in 15 cycles of PCR using primers Illumina 1.0 and Illumina 2.0. The libraries were quantified using Taqman, and pooled at a concentration of 10 pM. We performed paired-end sequencing of the libraries on the Genome Analyzer IIx using Illumina v4 sequencing chemistry.