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Next Generation Sequencing Facilitates Quantitative Analysis of FLox and SMYD2 KO Aortic Smooth Muscle Cell Transcriptomes

ABSTRACT: Purpose: Methyltransferase SMYD2 has been implicated in cancer cell growth and cardiomyocytes development. However, its role in vascular smooth muscle cell(VSMC) is still unknown. This study aims to investigate differentially expressed genes in Flox and SMYD2 knockout VSMC. Methods:RNA profiles of Aortic Smooth Muscle Cell isolated from Flox and SMYD2 knockout (SMYD2−/−) mice were generated by deep sequencing, in triplicate, using BGISEQ-500 platform. Firstly, we filter the low quality reads (More than 20% of the bases qualities are lower than 10),reads with adaptors and reads with unknown bases (N bases more than 5%)to get the clean reads. Then we map those clean reads onto reference genome, followed with novel gene prediction, SNP &INDEL calling and gene splicing detection. Finally, we identify DEGs (differentially expressed genes) between samples and do clustering analysis and functional annotations. Results: In our project, we sequenced 6 samples on BGISEQ-500 Platform in total and generated about 4.69 Gb per sample. The average genome mapping rate is 96.62% and the average gene mapping rate is 80.92%. 18,112 genes were identified in which 16,992 of them are known genes and 1,351 of them are novel genes. 13,680 novel transcipts were identified in which 11,251 of them are previously unknown splicing event for known genes, 1,351 of them are novel coding transcripts without any known features, and the remaining 1,078 are long noncoding RNA. Conclusions: Our study represents the first detailed analysis of SMYD2 KO VSMC transcriptomes, with biologic replicates, generated by RNA-seq technology.

ORGANISM(S): Mus musculus

PROVIDER: GSE161004 | GEO | 2023/05/12

REPOSITORIES: GEO

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Project description:We report two sets of potential circadian clock target genes by high-throughput transcriptome profiling (RNA-sequencing): one set from the corpus callosum of the adult mouse brain and the other set from adult neural stem cell cultured from the SVZ (neurospheres) of the mouse brain. First, the corpus callosum was dissected from three adult wild-type and two Bmal1 knockout (Bmal1-/-) mice, mRNA profiles were generated by deep sequencing using BGISEQ-500 RNA-Seq platform. The sequence reads that passed quality control were analyzed: we mapped more than 21 million total clean reads, more than 96% total Mapping Ratio, more than 86% Uniquely Mapping Ratio per sample to mouse genome. qRT-PCR validation with primers covering at least 3 known housekeeping genes and at least 5 well-known circadian clock genes were performed with SYBR Green assay. 103 differentially expressed genes (DEGs) were identified in the corpus callosum of Bmal1-/- mice (58 up-regulated and 45 down-regulated genes; |log2FC| > 1 & adjusted P-value < 0.05). Hypergeometric test revealed that potential functions of circadian clock target genes in the corpus callosum are secreted proteins as signaling molecules. For the second set of data, neurospheres were cultured from the SVZ of three adult wild-type and three adult Bmal1-/- mice, mRNA profiles were generated by deep sequencing using BGISEQ-500 RNA-Seq platform. The sequence reads that passed quality control were analyzed: we mapped more than 28 million total clean reads, more than 97% total Mapping Ratio, more than 68% Uniquely Mapping Ratio per sample to mouse genome. qRT-PCR validation with at least 3 known housekeeping genes and at least 5 well-known circadian clock genes using SYBR Green assay. 30 DEGs were identified in neurospheres cultured from Bmal1-/- mice (17 up-regulated and 13 down-regulated genes; |log2FC| > 1 & adjusted P-value < 0.05). Gene Set Enrichment Analysis (GSEA) revealed that potential circadian clock associated functions in the SVZ (neurospheres) are controlling diverse signal transductions.

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Next Generation Sequencing Facilitates Quantitative Analysis of FLox and SMYD2 KO Aortic Smooth Muscle Cell Transcriptomes

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