RNA-Seq of chicken TOC (replicate 1) 24 hours post CSW-1 ILTV inoculation
Libraries were prepared using a TrueSeq Stranded mRNA library preparation kit (Illumina Inc., San Diego, CA, USA) following the manufacturers recommendations. Briefly, poly-A containing mRNA, was isolated from 400 ng of total RNA from each replicate, using poly-T-oligo magnetic beads. Purified mRNA was then fragmented, and reverse transcribed to produce first strand cDNA using random primers and PCR amplification. This was followed by second strand cDNA synthesis using DNA polymerase I and RNase H. Prime ends of cDNA were then adenylated and subjected to single index adaptor ligation. The end products were purified and enriched by PCR for the generation of the final cDNA libraries. The quality of the cDNA libraries was assessed using the Agilent 4200 TapeStation system (Agilent Technologies, Santa Clara, CA, USA) and all samples conformed to the required fragment size (~260 bp) for sequencing. Libraries were then normalised to 1 nM, pooled, denatured and sequenced on a NextSeq500 sequencing platform (Illumina Inc., San Diego, CA,USA) using 80 bp paired-end approach