GSE97062
Next Generation Sequencing Facilitates The Study of the Effect of ABX464 in Intestinal Inflammation
Purpose: The goals of this study are to establish the protective effect of ABX464 in DSS-induced colitis comparing transcript levels between cells (BMDMs) ABX464-treated stimulated or not by LPS. Methods: 24 intestinal macrophages mRNA profiles of three distinct group of BMDM cultures from mice were generated by deep sequencing, using Illumina protocol. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: HISAT and Bowtie2 followed by genes expression quantification using a software package called RSEM. Results: Using an optimized data analysis workflow, we mapped about 45 million sequence reads per sample to the mouse genome (build mm9) and identified 1383 expressed genes in the LPS-stimulated BMDMs among which 439 and 397 genes increased and decreased in expression >= 1.5 log2-fold, respectively. Altered expression of some genes was confirmed with qPCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to inflammatory function. Data analysis with HISAT/Bowtie2 and RSEM workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the detailed analysis of BMDMs transcriptomes, with biologic samples, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Overall design: 24 intestinal mRNA profiles of three distinct group of BMDM cultures from mice were generated by deep sequencing, in simplicate, using Illumina platform.