Illumina HiSeq 2500 sequencing; Resistant and susceptible chicken lines show distinctive responses to Newcastle disease virus infection in the lung transcriptome
Resistant and susceptible chicken lines show distinctive responses to Newcastle disease virus infection in the lung transcriptome
Chickens were housed in 4 separate rooms (2 challenged rooms, 2 non-challenged rooms) and the two breeds were intermixed in each room in a biosafety level 2 facility. To obtain tissue samples at 2, 6, and 10 days post inoculation, approximately one-third of the birds were euthanized with sodium pentobarbital solution at each of the three time points. The lung was harvested and placed into RNAlater® solution (ThermoFisher Scientific, Waltham, Massachusetts) for short-term storage. Within a week, the lung tissue was removed from RNA-later and moved to -80°C freezer for long-term storage. Challenged chickens were inoculated with a high titered lentogenic Newcastle Disease Virus (NDV) strain via the eyes and nostrils at three weeks of age. Non-challenged chickens were given PBS via the same route. RNA was isolated from the lung tissue using the RNAqueous® kit (ThermoFisher Scientific, Waltham, Massachusetts) and samples were then DNase treated using the DNA-free™ kit (ThermoFisher Scientific, Waltham, Massachusetts). The quality of all samples was validated (RNA quality number>8.0) using the Fragment Analyzer™ (Advanced Analytical Technologies, Ankeny, Iowa).  A 500 ng input for each sample was utilized to construct the cDNA library using TruSeq® RNA Sample Preparation v2 Guide (Illumina, San Diego, California) using the high throughput protocol. Following cDNA library validation using the Fragment Analyzer™ (Advanced Analytical Technologies, Ankeny, Iowa).
Experimental Factor: infect
PBS control
Experimental Factor: breed
Leghorn
Experimental Factor: sampling time point
2