Confluent monolayers of polarized Calu-3 achieving stable transepithelial resistance were infected apically at a MOI of 1. After a 1-hour incubation, monolayers were washed to remove nonadherent virus and 2 ml of MEM-BSA was added to both apical and basolateral reservoirs of cells and left for the duration of the experiment.
Growth protocol
The human bronchial epithelial cell line Calu-3 was originally derived from subbronchial epithelium of a lung adenocarcinoma (ATCC) and cells were grown in Eagle's minimal essential medium (MEM) supplemented with 2 mM l-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1.5 g/liter sodium bicarbonate, and 10% fetal bovine serum. Calu-3 cells (5 × 105) were seeded onto Corning 24-mm-diameter semipermeable membrane inserts with a 0.4-μm pore size (Corning, NY) and cultured for 1 week with the addition of fresh culture medium every 2 to 3 days. Confluent monolayers of polarized Calu-3 achieving stable transepithelial resistance were infected apically at a MOI of 1. After a 1-hour incubation, monolayers were washed to remove nonadherent virus and 2 ml of MEM-BSA was added to both apical and basolateral reservoirs of cells and left for the duration of the experiment.
Extracted molecule
total RNA
Extraction protocol
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 microarray analyses.
Label
Cy3
Label protocol
The Agilent One-Color Microarray-Based Gene Expression Analysis Protocol was followed for the Cy3-cDNA probe preparation, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.
Hybridization protocol
The Agilent One-Color Microarray-Based Gene Expression Analysis Protocol was followed for all processing steps, including Cy3-cDNA probe preparation, hybridization and array washing.
Scan protocol
Dry slides were scanned on an Agilent DNA microarray scanner (Model G2505B) using the XDR setting.
Data processing
After control probes filtering : 58,717 probes including 8118 duplicated probes 3. After SUMMARIZATION OF non-CTRL PROBES: 50,599 probes 4. Filtering of probes: we require that the probes pass the following QC in at least 100% samples of 1 infected time-point: gIsFound, gIsWellAboveBG, gIsSaturated: no probe was excluded based on that criteria, gIsFeatNonUnifOL, and gIsFeatPopnOL. Probes passing qc: 29,382 probes only.
Transcriptomic characterization of the novel avian-origin influenza A (H7N9) virus: specific and intermediate host-response between avian (H5N1 and H7N7) and human (H3N2) viruses.
