Cells were infected with either mock virus preparation or ZEBOV at a multiplicity of infection (MOI) of 10.
Growth protocol
Cells were cultivated in RPMI 1640 (Invitrogen, Carlsbad, CA) containing 20% heat-inactivated human AB serum (Sigma-Aldrich, St. Louis, MO), penicillin (100 U/ml), streptomycin (100 µg/ml), and L-glutamine (2 mM); cells were incubated at 37 ºC in a humidified 5% CO2 environment
Extracted molecule
total RNA
Extraction protocol
Cell supernatants were removed from the cells 1 h or 6 h post infection, and RNA from cells was purified using the RNeasy Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions.
Label
biotin
Label protocol
Using 5 µg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (IDT, Coralville, IA). Double-stranded cDNA was purified by phenol-chloroform-isoamyl alcohol extraction and a standard ethanol precipitation. In the second step, the cDNA was converted to labeled cRNA (the target) using T7 RNA polymerase in the presence of biotinylated UTP and CTP (Enzo, Farmingdale, NY). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) and was followed by an ethanol precipitation of the labeled target. The target was fragmented to produce a uniform distribution of short cRNAs. 10µg each of fragmented target was combined with hybridization control oligomer (Affymetrix, Santa Clara, CA) and control cRNAs (1.5pM BioB, 5.0pM BioC, 25pM BioD, and 100pM Cre; Affymetrix, Santa Clara, CA), in hybridization buffer and applied to the array.
Hybridization protocol
The arrays were hybridized in a GeneChip Hybridization Oven 640 (Affymetrix, Santa Clara, CA) for 16 hours at 45°C, followed by washing, staining with streptavidin-phycoerythrin (SAPE; Molecular Probes, Eugene, OR), signal amplification with biotinylated anti-streptavidin antibody (Vector Labs, Burlingame, CA), and a final staining with SAPE. The staining and washing were performed using two GeneChip Fluidic Station 400s (Affymetrix, Santa Clara, CA) running standard Affymetrix fluidics script.
Scan protocol
The distribution of fluorescent material on the processed array was determined using the Agilent G2500 GeneArray Scanner (Agilent Technologies, Santa Clara, CA). The array image scan was processed using Affymetrix Microarray Suite, version 5.0 (MAS 5.0), which resulted in the generation of raw, probe level data in the CEL file format.
Description
Gene expression data from Ebola-infected macrophages
