cell were treated with 1 ug/ ml of endotoxin from Salmonella typhimurium-798 for 1, 2, 4 and 8 hours
Growth protocol
HD11 cells were cultured in RPMI 1640 medium (Sigma) supplemented with 10% heat-inactivated newborn calf serum, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES and 5 × 10−5 M 2-mercaptoethanol (pH 7.3) at 41 °C and 5% CO2.
Samples were labeled with the GeneChip® 3' IVT Express Kit (Affymetrix, part number 901229)
Hybridization protocol
The reagents for hybridization, wash and stain were also purchased from Affymetrix (Part No: 900720). The hybridization was conducted at 45oC for 16 hours in a GeneChip® hybridization oven 640 with constant rotation at 60 rpm. The arrays were washed and stained in the GeneChip® fludics station 450
Scan protocol
scanned with GeneChip® scanner 3000 7G. Affymetrix® GeneChip® Operation Software (GCOS) was used to control the fludics stations and scanner and to generate generic data (.DAT) files, probe cell intensity data (.CEL) files, and the Probe-level summarization (.CHP) files..
Description
SAMPLE 3 (2-0-1)(time-dose-rep)
Data processing
Data were normalized and expression measures computed using the Robust Multiarray Average (RMA) method (Irizarry et al., 2003), Bioconductor package 'affy.' A linear model with fixed effects for replications, endotoxin doses, times, and interactions between doses and times were fit to the expression data for each gene using the R package limma (Smyth, 2004, 2005). As part of each linear model analysis, P-values were obtained for the test for dose-by-time interaction, the test for changes over time within endotoxin dose groups, and the test for a dose effect at each time point. The P-values for each test were converted to q-values for false discovery rate estimation using the method of Nettleton et al. (2006).
