Next Generation Sequencing Facilitates Quantitative Analysis of wild type and Phlpp1-deficient Bone Marrow Derived macrophage Transcriptomes
Purpose: The purpose of this study is to detect activated or silenced genes during wild type (WT) and Phlpp1-deficient bone marrow derived macrophages (BMDMs). Gene expression differences between two samples could be found using transcriptome profiling (RNA-seq) analysis. Methods: Mouse BMDMs were generated from bone marrow cells in RPMI-1640 medium with recombinant mouse M-CSF (20ng/ml). BMDMs were stained to confirm the surface expression of CD11b and F4/80. Cells with purity >97.5% were used for subsequent experiments. WT and Phlpp1 deficient BMDMs were stimulated with IL-4 (20ng/ml) for 12 and 24 hours, of which RNA profiles were generated by deep sequencing, using Illumina. Results: We mapped about 10 million sequence reads per sample to the mouse genome, identified hundreds of genes with significant mRNA  between WT and Phlpp1 deficient BMDMs. Overall design: mRNA profiles of WT and Phlpp1 deficient BMDMs were generated by deep sequencing.