Next Generation Sequencing Transcriptomes Analysis of Irradiated mouse submandibular glands with or without transient activation of Hedgehog signaling

Purpose: To compare transcriptomes of nontreated (NT), Irradiated (IR and IR+GFP) and Hedgehog-activated (IR+Gli1 and IR+Shh) mouse submandibular glands (SMGs) Methods: SMG mRNA profiles of male adult NT mice or mice 7 days after local IR were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by HTseq. 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) and identified 20,743 transcripts in the SMGs with TopHat workflow. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Conclusions: Our study represents the first detailed analysis of SMG transcriptomes after IR with or without transient activation of Hedgehog (Hh) signaling, with biologic replicates, generated by RNA-seq technology. Our results show that IR impaired macrophages and related innate immune responses in SMGs, whereas transient Hh activation recovered them. Overall design: SMG mRNA profiles of male adult NT mice or mice 7 days after local IR were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500