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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE133482 | GEO | 2020/12/22

REPOSITORIES: GEO

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Project description:Purpose: We present a genome-wide investigation to distinguish mRNA substrates directly regulated by nonsense-mediated mRNA decay (NMD) from indirect secondary effects using true NMD null alleles Methods: mRNA profiles of wild type (N2), smg-1(r910), and smg-1(r910) smg-2(r915) animals were generated by deep sequencing, in triplicate, using Illumina HiSeq2000. We further performed deep sequencing in triplicate on RNAs that co-immunoprecipitate with the central effector of NMD, SMG-2, in both smg-1(r910) and smg-1(r910) smg-2(r915) mutant animals. The sequence reads that passed quality filters were analyzed at both the gene level and the intron level using Tophat2 and edgeR. 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 mm9) and identified 16,014 transcripts in the retinas of WT and Nrl−/− mice with BWA workflow and 34,115 transcripts with TopHat workflow. 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: We found that a significant portion of genes with increased expression in an NMD-deficient background are direct substrates of SMG-2. Among the list of direct targets, we find many mRNAs from pseudogenes, alternative splice isoforms harboring PTCs, mRNAs encoding RNA processing factors, and two recently expanded gene families are directly regulated by the NMD pathway.

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

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