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Next-Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Kdm2b-/- Peritoneal Macrophage Transcriptomes

ABSTRACT: Purpose: The purpose of this study is to detect activated or silenced genes during Kdm2b-deficient peritoneal macrophages and the control cells. Gene expression differences between two samples could be found using transcriptome profiling (RNA-seq) analysis. Methods: Mouse peritoneal macrophages were harvested from mice 4 days after thioglycollate (BD, Sparks, MD) injection. Macrophages 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 variation between Kdm2b-deficient macrophages and the control cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE120651 | GEO | 2019/12/31

REPOSITORIES: GEO

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Project description:Purpose: Previous work has demonstrated that miR-33 is an important regulator of lipid metabolism and atherogenesis. By performing bone marrow transplant experiments into LDLR-/- mice, our work demonstrates that the effects of miR-33 in macrophages play a major role in its ability to reduced atherosclerotic plaque burdon. To have performed extensive additional characterization of the effects of miR-33 deficiency in macrophages icluding RNA-seq analysis of peritoneal macrophages from wildtype, miR-33-/-, LDLR-/-, and miR33-/-/LDLR-/- animals. Methods: Thioglycolate elicited peritoneal macrophages from WT and miR-33-/- mice were harvested by peritoneal lavage. Cells were then plated for 2hr, then washed to remove non-adherant cells. Macrophages were then scraped, pelleted and frozen at -80◦C. Total RNA from WT and miR-33-/- thioglycollate-elicited peritoneal macrophages was extracted and purified using a RNA isolation Kit (Qiagen) followed by DNAse treatment to remove genomic contamination using RNA MinElute Cleanup (Qiagen). The purity and integrity of total RNA sample was verified using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). rRNA was depleted from RNA samples using Ribo-Zero rRNA Removal Kit (Illumina). RNA libraries from WT BMDMs were performed TrueSeq Small RNA Library preparation (Illumina) and were sequenced for 45 cycles on Illumina HiSeq 2000 platformm (1 x 75bp read length). The reads obtained from the sequencer are trimmed for quality using in-house developed scripts. The trimmed reads are aligned to the reference genome using TopHat2. The transcript abundances and differences calculated using cuffdiff. The results were plotted using R and cummeRbund using in-house developed scripts. Results: Our RNA-seq analysis has allowed us to identify genes and pathways that are altered in miR-33 deficient peritoneal macrophages under hyperlipidemic conditions (LDLR-/- vs. miR33-/-/LDLR-/-). Further analysis of gene expression changes that occur between wildtype and LDLR-/- animals has allowed us to identify which of these changes are likely due to differences in lipid loading and which are independent of these effects.

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Next-Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Kdm2b-/- Peritoneal Macrophage Transcriptomes

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