title: RNA-seq of pigeon breast muscle
design: Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according  to the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer  (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel  electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT)  beads, while prokaryotic mRNA was enriched by removing rRNA by Ribo-ZeroTM Magnetic  Kit (Epicentre, Madison, WI, USA). Then the enriched mRNA was fragmented into short  fragments using fragmentation buffer and reverse transcripted into cDNA with random  primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and  buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit(Qiagen,  Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing  adapters. The ligation products were size selected by agarose gel electrophoresis, PCR  amplified, and sequenced using Illumina HiSeq2509 by Gene Denovo Biotechnology Co.  (Guangzhou, China).