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RNA-seq of dexamethasone-treated mouse primary hepatocytes infected with shControl or shSETDB2 adenovirus

ABSTRACT: Purpose: The goal of this study is to identify differentially expressed genes in SETDB2 deficient mouse primary hepatocytes (MPH) treated with dexamethasone when compared to control MPH Methods: Total RNA from MPH treated with either shControl or shSETDB2 adenovirus (pool of 3 mice per group) was isolated by the TRIzol method (Life Technologies) according to manufacturer’s instructions. Total RNA was DNase-treated and RNA integrity was evaluated by Biolanalyzer (Agilent). RNA sequencing was performed by the SBP Genomics Core Facility in Lake Nona (Orlando, FL) using the Illumina HiSeq platform and the following parameters: paired-end read length: 100 BP; number of output reads: 300m reads. The Tophat+Cufflinks pipeline was used for the RNA-seq analysis as described previously (Haas et al., 2013; Trapnell et al., 2012). Differentially expressed genes in shSETDB2 vs. shControl MPH were determined using a 2-fold cutoff, p-value of less than 0.05, and FPKM (fragments per kilobase of transcript per million mapped reads) cutoff of 1. The shSETDB2 and shControl RNA-seq reads were mapped to the mm10 genome using Tophat (Trapnell et al., 2009). The mapping results were further analyzed by Cufflinks, which outputs the final differential gene list.

ORGANISM(S): Mus musculus

PROVIDER: GSE85278 | GEO | 2016/08/28

SECONDARY ACCESSION(S): PRJNA338065

REPOSITORIES: GEO

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Project description:Purpose: Cucumber (Cucumis sativus L.) is an economically important vegetable crop worldwide, and cucumber fruit spine density has an important impact on the commercial value. However, little is known about the regulatory mechanism for the fruit spine formation.In this study, the transcriptome analyses of ovaries and pericarps from numerous-spine parent and few-spine parent were conducted to identify the gene regulatory networks involved in the formation and development of numerous fruit spines in cucumber. Methods: Cucumber mRNA profiles of ovaries and pericarps from numerous-spine parent and few-spine parent were generated by deep sequencing, in triplicate, using Illumina HiSeq 4000. Then, clean data (clean reads) were obtained by removing reads containing adapters, reads containing poly-N sequences and low-quality reads from the raw data. Simultaneously, the Q20, Q30 and GC contents of the clean data were calculated. All of the downstream analyses were based on the high-quality clean data. Clean paired-end reads were mapped to the reference genome using TopHat v2.0.12 (Trapnell et al. 2012). Then, the FPKM (fragments per kilobase of transcript sequence per million base pairs sequenced) value of each gene was calculated to estimate gene expression levels (Trapnell et al. 2010). Genes with an adjusted P-value < 0.05 identified by DESeq were assigned as differentially expressed genes(DEGs). Results: We generated 42.96-57.53 million raw reads from each library, and 39.85-54.02 million clean reads were obtained after the removal of low-quality reads and adapter sequences. Among the clean reads, 79.03-80.94% were mapped to the gene database . Based on the KEGG database, pathway enrichment analysis was performed to identify significantly enriched metabolic pathways or signal transduction pathways in DEGs. Plant hormone signal transduction was significantly enriched in up-regulated genes in both F_6DBF compared with M_6DBF and F_0DAA compared with M_0DAA. Conclusions: Based on the transcriptome analysis, we excavated possible biological regulatory networks involved in the formation and development of numerous fruit spines in cucumber. This work will promote the exploration of molecular mechanisms that regulate cucumber fruit spine density.

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RNA-seq of dexamethasone-treated mouse primary hepatocytes infected with shControl or shSETDB2 adenovirus

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