GSE129355
RNA Sequencing Analysis of Wild Type and ColcreER; Tbx4flox/flox Whole Lung and Mesenchymal Extracellular Vesicle Transcriptomes
Purpose: The goals of this study are to compare wild type (WT) and ColcreER; Tbx4flox/flox whole lung to lung mesenchymal extracellular vesicle (EV) trasncriptome profilling by RNA sequencing (RNA-seq) and to analyze the differentiated genes. Methods: Total RNA was isolated from WT and ColcreER; Tbx4flox/flox whole lung and cultured lung mesenchymal extracellular vesicle. mRNA profiles were generated by deep sequencing, using Illumina HiSeq 3000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl-/- mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl-/- retina, with a fold change =1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, 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 retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study analyzed transcriptomes of WT and ColcreER; Tbx4flox/flox whole lungs and cultured lung mesenchymal extracellular vesicle, with biologic replicates, generated by RNA-seq technology. This study provides a framework for the application of transcriptome profiling towards characterization of differentiated genes in whole lung and mesenchymal extracellular vesicle of WT and Tbx4creER; Ghrflox/flox mice . Overall design: lung and extracellular vesicle mRNA profiles of adult wild type (WT) and Tbx4creER; Ghrflox/flox mice were generated by deep sequencing using Illumina HiSeq 3000.