GSE133159
Next Generation Sequencing Facilitates Quantitative Analysis of self-reactive B cells Transcriptomes in PPs
Purpose: The purpose of this study is to identify novel regulator of B cell tolerance to gut associated self antigens using HEL system. The goals of this study are to compare NGS-derived (RNA-seq) differentially expressing genes in antigen encountered self-reactive B cells to non-antigen expirenced cells from different tissues. Methods: Self-reactive (HEL binding) cells were sorted from the spleen, mesenteric lymph nodes and Peyer's Patches of VillinCre-mDelloxp-SWHEL mice. RNA was isolated and cDNA librabries were made using SMART (Switching Mechnaism at 5' End RNA template) technology followed by NGS using Nextera from Illumina. 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. qRT–PCR validation was performed using SYBR Green assays Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm10). 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 represents the first detailed analysis of self-reactive B cells in gut transcriptomes, with biologic and technical replicates, 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 or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Overall design: Self-reactive B cells in gut transcriptomes