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Next Generation Sequencing Facilitates Quantitative Analysis of shControl and shLINC00205 Gastric Cancer Cell Lines Transcriptomes

ABSTRACT: Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare the transcriptome differences between knockdown LINC00205 and negative control gastric cancer cells. Methods: Total RNA from the BGC823 cells with LINC00205 knockdown and negative control were isolated using RNeasy mini kit (Qiagen, Germany). Paired-end libraries were synthesized by using the TruSeq RNA Sample Preparation Kit (Illumina, USA) following TruSeq RNA Sample Preparation Guide. Briefly, The poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. Purified libraries were quantified by Qubit 2.0 Fluorometer (Life Technologies, USA) and validated by Agilent 2100 bioanalyzer (Agilent Technologies, USA) to confirm the insert size and calculate the mole concentration. Cluster was generated by cBot with the library diluted to 10 pM and then were sequenced on the Illumina HiSeq X-ten (Illumina, USA). The library construction and sequencing was performed at Shanghai Biotechnology Corporation. STAR (version:2.7.6a) was used to map the cleaned reads to the human GRCh38 reference genome with two mismatches 1. Then ,we ran Subread/featureCounts (version:2.0.2) with a reference annotation to generate gene expression counts and FPKM values for known gene model. Results: We identified a set of differentially expressed genes after LINC00205 knockdown. The result of GO and KEGG enrichment analysis showed high confidence of genes enriched in the function of cell proliferation, cell cycle, and cell adhesion, consistent with the canonical molecules of these pathways significantly modulated. Conclusion:The results suggested that LINC00205 could affect the key regulated pathway that controls the progression and metastasis of GC.

ORGANISM(S): Homo sapiens

PROVIDER: GSE186732 | GEO | 2024/10/28

REPOSITORIES: GEO

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Project description:Purpose: Several members of the tripartite motif (TRIM) family of E3 ubiquitin ligases regulate immune pathways including the antiviral type I interferon (IFN-I) system. Therefore, we aimed to investigate the mechanism of IFN-I regulation upon West Nile virus infection using TRIM6 knockout (TRIM6-KO) human airway epithelial cells (A549). Methods: RNAs were extracted from wild-type (WT) and TRIM6KO A549 cells infected with WNV or mock-treated. RNA quality was assessed by visualization of 18S and 28S RNA bands using an Agilent BioAnalyzer 2100 (Agilent Technologies, CA); the electropherograms were used to calculate the 28S/18S ratio and the RNA Integrity Number. Poly-A+ RNA was enriched from total RNA (1 μg) using oligo dT-attached magnetic beads. First and second strand synthesis, adapter ligation, and amplification of the library were performed using the Illumina TruSeq RNA Sample Preparation kit as recommended by the manufacturer (Illumina, Inc). Library quality was evaluated using an Agilent DNA-1000 chip on an Agilent 2100 Bioanalyzer. Quantification of library DNA templates was performed using qPCR and a known-size reference standard. Cluster formation of the library DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation using conditions recommended by the manufacturer. Paired end 50 base sequencing by synthesis was performed using TruSeq SBS kit v3 (Illumina) on an Illumina HiSeq 1500 using protocols defined by the manufacturer. The alignment of NGS sequence reads was performed using the Spliced Transcript Alignment to a Reference (STAR) program, version 2.5.1b, using default parameters. We used the human hg38 assembly as a reference with the UCSC gene annotation file. The –quantMode GeneCounts option of STAR provided read counts per gene, which were input into the DESeq2 (version 1.12.1) differential expression analysis program to determine expression levels and differentially expressed genes. Processed data are presented as reads per kilobase transcript per million (RPKM). Results: Using next generation sequencing, we identified VAMP8 as a factor involved in this TRIM6-mediated antiviral response. In support, VAMP8 knockdown resulted in reduced Jak1 and STAT1 phosphorylation and impaired induction of several ISGs following WNV infection or IFNβ treatment. Conclusions: Our results provide evidence that TRIM6 contributes to the antiviral response against WNV and identified VAMP8 as a novel regulator of the IFN-I system

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Next Generation Sequencing Facilitates Quantitative Analysis of shControl and shLINC00205 Gastric Cancer Cell Lines Transcriptomes

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