Data from: Transcriptional profiling of lung macrophages following ozone exposure in mice identifies signaling pathways regulating immunometabolic activation

Description

Notes

Methods

Total RNA was extracted as described above from 3 mice/treatment group. In a pilot study, we found that 3 mice were sufficient to identify a significant difference in Ptgs2 gene expression by qPCR at α = 0.05 and power = 80%. RNA integrity numbers (RINs) were confirmed to be ≥ 8.8 using a 2100 Bioanalyzer Instrument (Agilent, Santa Clara, CA). cDNA libraries were prepared using mouse TruSeq® Stranded Total RNA Library Prep kit (illumina, San Diego, CA) and quantified using a KAPA Library Quantification kit (Roche, Pleasanton, CA). cDNA libraries were sequenced (75 bp single-ended, ~35-44M reads per sample) on an Illumina NextSeq instrument. Raw reads in FastQ files were trimmed using Trimmomatic-0.39 (Bolger et al. 2014) and quality control of trimmed files performed using FastQC. Salmon was used to align reads in mapping-based mode with selective alignment against a decoy-aware transcriptome generated from mouse transcriptome GENCODE Release M23 (GRCm38.p6). Estimated counts per transcript were generated using the gcBias flag and normalized to transcript length to correct for potential changes in gene length across samples from differential isoform usage (Love et al. 2016; Patro et al. 2017). Transcript level quantitation data were aggregated to the gene-level using tximport (Soneson et al. 2015). Differential gene expression analysis was performed with air exposed mice as controls using DESeq2 with corrections for differences in library size (Love et al. 2014) in R version 4.0.3. Significantly enriched canonical pathways and upstream regulators were identified with Ingenuity IPA Version 65367011 (QIAGEN Inc, https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/) using a right-tailed Fisher's Exact Test (Krämer et al. 2014). A less stringent criteria (fold change > 1.3 and experimental false discovery rate [padj] < 0.05) was used to augment the number of genes included in the pathway analysis (Bennett et al. 2024). Data were deposited NCBI's Gene Expression Omnibus (Edgar et al. 2002) and are accessible through GEO Series accession number GSE237594 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE237594).