RNA-Seq of allantoic fluid harvested from Beau-CK infected chicken eggs
Virus infected allantoic fluid was clarified by centrifuging at 11200 xg for 10 minutes and the supernatant then subjected to ultracentrifugation at 50,000 rpm for 1 hour on a 30% sucrose cushion and the pellet was resuspended in RTL lysis buffer (Qiagen, Gaithersburg, MD).  Total RNA was checked for quality using a Bioanalyser 2100 RNA Pico kit (Agilent, San Diego, CA)) and quantitated using a Qubit (Life technologies) and normalised for a 100ng input.  Sequencing library preparation was performed using a NEBNext directional Ultra RNA-Seq kit (NEB, Ipswich, MA).  Library QC was performed using the Bioanalyser 2100 DNA 1000 kit and Qubit, prior to pooling.  The library pools were quantified using a NEBNEXT Illumina library quantitation kit (NEB) before being diluted and loaded onto a single lane of an Illumina HiSeq 4000 (Illumina, San Diego, CA).