Transcriptomic analysis of peritoneal macrophages from MED1 fl/fl and macrophage MED1 KO mice

Purpose: The goal of this study is to obtain the differential gene expression in MED1fl/fl and MED1ΔMac peritoneal macrophages (PMs) stimulated with or without LPS. Methods: Pooled PMs were collected from 8-week-old MED1fl/fl and MED1ΔMac mice (n=6 mice/group), and then treated without or with lipopolysaccharide (LPS; 50 ng/mL) for 6 hours. PMs mRNA profiles were generated by deep sequencing, in duplicate, using Illumina NextSeq500. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: reads were mapped to the mm9 whole genome using tophat and RPKM were calculated using cufflink. qRT–PCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 12 million sequence reads per sample to the mouse genome (build mm9) and identified 24,130 transcripts in the PMs of MED1fl/fl and MED1ΔMac mice. Data analysis with TopHat and Cufflinks workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the detailed analysis of PMs transcriptomes, with biologic replicates, generated by RNA-seq technology. Our results show that the expression of more than 20 genes involved in innate immune response and M1 polarization was significantly higher in MED1ΔMac than in MED1fl/fl macrophages after LPS treatment. Upregulated genes in MED1ΔMac macrophages are the proinflammatory genes. PMs mRNA profiles of 8-week-old MED1fl/fl and MED1ΔMac mice