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RNA-Seq analysis, transcriptome assembly and gene expression profile analysis for vascular smooth muscle cells with up- or downregulation of CKLF1

ABSTRACT: To understand the role of Cklf1 in the proliferation of vascular smooth muscle cells (VSMC), we quantified the whole genome transcriptomes of VSMC that were treated by Cklf1 adenovirus (n=3) or control (n=3) for 24 h using RNA-seq. In total, we obtained over 82 million clean reads from each library after trimming adaptor sequences, low quality reads and multiple mapped reads. The clean reads were mapped to 29330 genes annotated in the rat reference genome (Rattus_norvegicus 6.0). We then identified differentially expressed genes (DEGs) between Cklf1-associated adenovirus and controls by comparing RNA-Seq data between the two groups. A total of 238 DEGs for Ad-GFP vs. Ad-Cklf1 and 468 DEGs for shRNA Scramble vs. shRNA Cklf1 were defined using the thresholds of FDR ≤ 0.05, difference ratio of FPKM (fragments per kilobase of exon model per million mapped fragments) ≥ 2 and diverge probability ≥ 0.8. The increased CKLF1 resulted in 34 up-regulated and 204 down-regulated genes while knockdown of Cklf1 led to 358 up-regulated and 110 down-regulated genes.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE111398 | GEO | 2021/03/05

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 analysis, transcriptome assembly and gene expression profile analysis for vascular smooth muscle cells with up- or downregulation of CKLF1

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