Next Generation Sequencing Facilitates Quantitative Analysis of Spermidine Inhibited Transcriptomes

Purpose: Spermidine inhibits the activation of interferon primed dendritic cells. The goals of this study are to identify genes and pathways invovled in the regulation. Methods: Mouse monocyte derived dendritic cells were primed with interferon α for 24 hours, and then cultured with R837, together with or without spermidine for 18 hours. Then, the next-generation libraries of mRNA were prepared using VAHTS mRNA-seq v2 Library Prep Kit for Illumina® (Vazyme, Nanjing, China). The Library quality was determined by Bioanalyzer 4200 (Agilent, Santa Clara, CA, USA). Then the mRNA-seq libraries were sequenced in HiSeq X10 system (Illumina, San Diego, CA, USA) on a 150bp paired-end run. The differentially expressed genes were selected as having more than 1 fold difference in their geometrical mean expression between the compared groups and a statistically significant p-value (<0.05) by analysis of DEseq2. The GO analysis on differentially expressed genes was performed with an R package: Clusterprofiler using a p<0.05 to define statistically enriched GO categories. Pathway analysis was used to determine the significant pathway of the differential genes according to Kyoto Encyclopedia of Genes and Genomes Database (http://www.genome.jp/kegg/) and DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/). Results: Genes in the JAK/STAT pathway and the NF-κB pathway were inhibited by spermidine. As a result, the pro-inflammatory cytokines including IL-1β, IL-12a, IL-12b (IL-12/23 p40), IL-6 and IL-23a decreased obviously upon spermidine treatment MRNA profiles of Control, R837 stimulated and R837 plus Spermidine treated moDCs were generated by deep sequencing, in triplicate, using illumina HiSeq X10 system