Cells were seeded in 6-well plates (1 x 10e6 cells/well) two days prior to infection. Immediately preceding infection, cell monolayers were washed with fresh medium and inoculated with either SARS viruses or A/CA/04/2009 at an MOI of 2 and subsequently incubated at 37°C for 40 minutes. Mock-infected controls were inoculated with culture medium only. Following the incubation, cell monolayers were washed 3X with 1X PBS and fresh medium was added to the wells prior to time 0.
Growth protocol
Human airway epithelium cultures (HAE) were generated by provision of an air-liquid interface for 6 to 8 weeks to form well-differentiated , polarized cultures that resemble in vivo pseudo-stratified mucociliary epithelium.
Extracted molecule
total RNA
Extraction protocol
At 0, 12, 24, 36, 48, 60, 72, 84 and 96 hours post-infection (hpi) (SARS-CoV virus), 0, 24, 48, 60, 72, 84 and 96 hours post-infection (hpi) (SARS ddORF6 and BatSRBD viruses) or 0, 6, 12, 18, 24, 36 and 48 hpi (H1N1) triplicate/quadruplicate wells of infected cells or mock infected were washed with 1X PBS and lysed directly with 1 ml of Trizol (Invitrogen) according to the manufacturer’s recommendation. The resulting lysates were stored at -80°C until further processing. All Trizol lysates were processed simultaneously: they were phase-separated, and RNA was isolated from the aqueous phase (diluted 2 fold with RLT buffer) using Qiagen RNeasy Mini columns and the manufacturer’s recommended protocol (Qiagen Inc., Valencia, CA). RNA quality was assessed on an Agilent 2100 Bioanalyzer using the nanochip format, and only intact RNA was used for quantitative PCR (qPCR) and microarray analyses.
Label
Cy3
Label protocol
The Agilent One-Color Microarray-Based Gene Expression Analysis Protocol was followed for the Cy3-cDNA probe preparation.
Hybridization protocol
The Agilent One-Color Microarray-Based Gene Expression Analysis Protocol was followed for hybridization and array washing. Two hundred fifty ng of each RNA sample was hybridized to one Agilent 4X44K human HG (Design ID 014850) array.
Scan protocol
Dry slides were scanned on an Agilent DNA microarray scanner (Model G2505B) using the XDR setting.
Data processing
Raw images were analyzed using the Agilent Feature Extraction software (version 9.5.3.1) and the GE1-v5_95_Feb07 extraction protocol. All arrays were required to pass Agilent QC flags. Extracted raw data were background corrected using the norm-exp method and quantile normalized using Agi4x44PreProcess and RMA Bioconductor packages.
