Next Generation Sequencing Facilitates Quantitative Analysis of Different Transcriptomes betwwen AA, AG, GG pDCs

Purpose: NCF1 gene single nucleotide polymorphism (SNP) rs201802880 (G to A change) promotes the activation of plasmacytoid dendritic cells. The goals of this study are to identify genes and pathways invovled in the regulation. Methods: Mouse plasmacytoid dendritic cells isolated from GG, AG or AA allele mice, were stimulated with R848 for 12 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 TLR7-IRF7 pathway were augmented by NCF1 SNP. As a result, the production of type I IFNs increased in AA pDCs. MRNA profiles of R848 stimulated pDCs with different alleles were generated by deep sequencing, in triplicate, using illumina HiSeq ⅹ10 system