7 protocols
AccessionType
normalization data transformation protocol
Data pre-processing and filtering was done using Partek software, v.6.6 and included: RMA background correction, quantile normalisation across all chips in the experiment, log2 transformation, median polish summarisation.
array scanning and feature extraction protocol
After washing and staining, the arrays were scanned with the Affymetrix GeneChip Scanner 3000 7G. Gene-level expression signal estimates were derived from CEL files generated from raw data using the multi-array analysis (RMA) algorithm implemented from the Affymetrix GeneChip Command Console Software Version 3.0.1.
nucleic acid hybridization to array protocol
Array hybridisation was performed according to the manufacturer's instructions (Affymetrix). Labeled samples were hybridized to the Affymetrix Chicken Gene 1.0 ST Arrays in a GeneChip Hybridization Oven for 16 h at 45°C and 60 rpm.
nucleic acid labeling protocol
Labeling of samples for array analysis was performed using the GeneChip WT Terminal labeling assay (Affymetrix) with 100 ng input RNA.
treatment protocol
Recombinant chicken IFNα was prepared as previously reported (Laidlaw et al, 2013) and was added in culture media to a final concentration of 1000 u/ml. Confluent cells were treated with chicken IFNα or mock treated and incubated for six hours before harvesting. Cells were stored at -80°C in RNAlater (Sigma) until RNA extraction. The experiment was repeated in triplicate with three different batches of CEFs.
nucleic acid extraction protocol
Total RNA was extracted from cells using an RNeasy kit (Qiagen) according to the manufacturer’s instructions. On-column DNA digestion was performed using RNase-free DNase (Qiagen) to remove contaminating genomic DNA. RNA samples were quantified using a Nanodrop Spectrophotometer (Thermo Scientific) and checked for quality using a 2100 Bioanalyzer (Agilent Technologies). All RNA samples had an RNA integrity number (RIN) ≥ 9.8.
growth protocol
Freshly isolated CEFs were provided by the Institute of Animal Health, Compton, Berks, UK. Cells were seeded in T25 flasks (Greiner Bio One; 5.6 x 106 cells/flask) and cultured overnight in 5.5 ml 199 media (Gibco®, Invitrogen) supplemented with 8% heat-inactivated newborn calf serum (NBCS; Gibco®, Invitrogen), 10% tryptose phosphate broth (TPB; Sigma), 2% nystatin (Sigma) and 0.1% penicillin streptomycin (Gibco®, Invitrogen).