GSE160489
Next Generation Sequencing Facilitates Quantitative Analysis of basic diet-treated (CON),lipopolysaccharide-treated(LPS), or lipopolysaccharide+genistein treated broiler chicks (GEN) thymic Transcriptomes
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of biological process. The goals of this study are to characterize the effects of genistein on the thymic transcriptome of chicks, when suffering from lipopolysaccharide challenge using RNA-Seq technology Methods: Thymic mRNA profiles of 21-day-old chicks with basic diet and lipopolysaccharide-treated, or lipopolysaccharide+genistein treated chick mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. 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 TaqMan and SYBR Green assays Results: Using an optimized data analysis workflow, we mapped about 70 million sequence reads per sample to the chicken genome and identified 25,016 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 6 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. We identified 712 DEGs (367 upregulated and 345 downregulated) between the LPS vs CON group with a |fold change| >1 (P =0.05, Figure 4 a). There are 1926 DEGs (1014 upregulated and 912 downregulated) in the GEN group compared with the LPS groupAltered expression of 6 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. Conclusions: Our research has evidenced a novel finding in chicks that dietary genistein could inhibit the increased percentage of CD3+ T lymphocytes, and CD4+/CD8+ T lymphocyte ratio in the peripheral blood induced by lipopolysaccharide injection. Furthermore, dietary genistein exposure altered the mRNA expression profile and AS signatures in the thymus, and regulated the immune response, along with the improvement of thymus index and apoptotic index after lipopolysaccharide challenge. It would help to understand the role of dietary genistein to the regulation of immune function and provide theoretical support for improving healthy production of poultry. Overall design: Thymic mRNA profiles of 21-day old basic diet-treated, lipopolysaccharide-treated, or lipopolysaccharide+genistein treated chicks