Human monocyte-derived macrophages (hMDMs) were separated from fractions of peripheral blood mononuclear cells (PBMCs) obtained from the blood of healthy donors using a lymphocyte separation medium density gradient. Macrophages were treated with the Newman strain of S. aureus at an MOI of 1:50 for 8, 24, and 48 hours at 37C. Untreated macrophages serve as controls.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with the Qiagen RNeasy kit according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 ug total RNA (Expression Analysis Technical Manual, 2004, Affymetrix).
Hybridization protocol
cRNA were hybridized on Affymetrix GeneChip Human Genome U133 Plus 2.0 Array according to the standard Affymetrix protocol (Expression Analysis Technical Manual, 2004, Affymetrix). GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Scan protocol
GeneChips were scanned using an Affymetrix scanner.
Description
SA-treated hMDMs
Data processing
Affymetrix GeneChip Operating Software (GCOS v1.0, http://www.affymetrix.com) was used to perform the preliminary analysis of the chips. All *.cel files, representing individual replicates, were scaled to a trimmed mean of 500 to produce the *.chp files. A pivot table with all samples was created including calls, call p-value and signal intensity for each gene.
