Genechip mouse gene st 2.0 arrays

RNA was isolated by Rneasy kit (Qiagen, 74106, Hilden, Germany). To purify RNA, Rnase-Free Dnase Set was used (Qiagen, 79254). RNA integrity was measured using the Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). A 100 ng aliquot of total RNA was linearly amplified. Then, 5.5 µg of cDNA was labeled and fragmented using the GeneChip WT PLUS reagent kit (Affymetrix, 902280, Santa Clara, CA, USA) following the manufacturer’s instructions. Labeled cDNA targets were hybridized to Affymetrix GeneChip Mouse Gene ST 2.0 arrays for 16 h at 45 °C rotating at 60 rpm. The arrays were washed and stained using the Fluidics Station 450 (Thermo Fisher, 00-0079, Waltham, MA) and scanned using a GeneChip Scanner 3000 (Thermo Fisher, 00-0210). Signal intensities were quantified by Affymetrix Expression Console version 1.3.1 (Thermo Fisher). Background correction and quantile normalization were performed to adjust for technical bias, and probe-set expression levels were calculated by the RMA method. After filtering above noise cutoff (p < 0.01), there were 9528 probe-sets tested by linear model. A variance smoothing method with fully moderated t-statistic was employed for this study and was adjusted by controlling the mean number of false positives. QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA) was used to identify pathways that were significantly affected by treatments.

Total RNA was isolated from alveolar macrophages as described above and quality assessed by using an Agilent Bioanalyzer. Affymetrix GeneChip Mouse Gene ST 2.0 arrays were used to determine transcriptomic profiles. Microarray analysis and quantitative assessment were performed by the Boston University Medical Campus Microarray and Sequencing Resource. CEL files were normalized to produce gene-level expression values using the implementation of the Robust Multiarray Average (RMA) [32 (link)] in the affy package (version 1.36.1)[33 (link)] included within in the Bioconductor software suite (version 2.11) and an Entrez Gene-specific probeset mapping (version 17.0.0) from the Molecular and Behavioral Neuroscience Institute (Brainarray) at the University of Michigan. Array quality was assessed by computing Relative Log Expression (RLE) and Normalized Unscaled Standard Error (NUSE) using the affyPLM Bioconductor package (version 1.34.0) [34 ]. Moderated t tests were performed using the limma package (version 3.14.4). Correction for multiple hypothesis testing was accomplished using the Benjamini-Hochberg false discovery rate (FDR) [35 ]. Analyses were performed using the R environment for statistical computing (version 2.15.1). Raw data are available at the NCBI Gene Expression Omnibus archive with the accession GSE98222.