Project description:We report the application of ChIP-Seq technology for analyzing the DNA binding sites of SOD1 in the nucleus of HeLa cells. By obtaining a plenty of sequence from chromatin immunoprecipitated DNA, we generated genome-wide DNA binding sites of SOD1. After sequencing of ChIP samples, 42,737,195, 49,950,032, and 38,825,768 clean reads for control group, H2O2 treated group and LD100 (a specific inhibitor of SOD1) treated group were obtained through trimming the raw reads. We find that SOD1 occupies DNA sites with distinct sequence preference in the nucleus. The treatment with either H2O2 or LD100 was found to decrease the strength of SOD1 binding to DNA, indicating that the H2O2 exposure- or SOD1 inhibition-mediated redox dyshomeostasis may result in decreased genes that are reasonably regulated through alteration of SOD1 structures compared to control.
Project description:Purpose: The goal of this study is to compare NGS-derived wild type and Hnrnpul1 knockout (Hnrnpul1-/-) RAW 264.7 cells transcriptomes with or without LPS stimulation. Methods: Sequancing was performed by Novogene China Co. Ltd. RNA profiles of wild type and Hnrnpul1-/- RAW 264.7 cells as well as LPS stimulated (10 h) wild type and Hnrnpul1-/- RAW 264.7 cells were generated by deep sequencing using Illumina Novaseq 6000. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. Method of TMM was used to normalize the readcount. Negative binomial distribution model was used to calculate the P value, and FDR was calculated by the method of Benjaminiand Hochberg. Results: Using an optimized data analysis workflow, we mapped about 40 million sequence reads per sample to the mouse genome (GRCm38/mm10). Comparing to wild type RAW 264.7 cells, 237 genes were up-regulated and 181 genes were down-regulated in Hnrnpul1-/- cells. At 10 h following LPS stimulation, 341 genes were up-regulated and 288 genes were down-regulated in Hnrnpul1-/- cells. Genes were pre-ranked according to log2FoldChange(KO/WT) followed by GSEA and 6 gene sets were significantly enriched. Significantly differential genes were undergone GO analysis (biological process) and biological process including cell-cell adhesion, positive regulation of cell activation and regulation of response to external stimulus were enriched. Conclusions: Lacking Hnrnpul1 promotes the expression of inflammatory cytokines in LPS stimulated RAW 264.7 cells.
Project description:Vesicular stomatitis virus (VSV) has shown considerable promise both as an immunization vector and as an oncolytic virus. In both applications, an important concern is the safety profile of the virus. To generate a highly attenuated virus, we added two reporter genes to the 3' end of the VSV genome, thereby shifting the NPMGL genes from positions 1 to 5 to positions 3 to 7. The resulting virus (VSV-12'GFP) was highly attenuated, generating smaller plaques than four other attenuated VSVs. In one-step growth curves, VSV-12'GFP displayed the slowest growth kinetics. The mechanism of attenuation appears to be due to reduced expression of VSV genes downstream of the reporter genes, as suggested by a 10.4-fold reduction in L-protein RNA transcript. Although attenuated, VSV-12'GFP was highly effective at generating an immune response, indicated by a high-titer antibody response against the green fluorescent protein (GFP) expressed by the virus. Although VSV-12'GFP was more attenuated than other VSVs on both normal and cancer cells, it nonetheless showed a greater level of infection of human cancer cells (glioma and melanoma) than of normal cells, and this effect was magnified in glioma by interferon application, indicating selective oncolysis. Intravenous VSV-12'GFP selectively infected human gliomas implanted into SCID mice subcutaneously or intracranially. All postnatal day 16 mice given intranasal VSV-12'GFP survived, whereas only 10% of those given VSV-G/GFP survived, indicating reduced neurotoxicity. Intratumoral injection of tumors with VSV-12'GFP dramatically suppressed tumor growth and enhanced survival. Together these data suggest this recombinant virus merits further study for its oncolytic and vaccine potential.
Project description:Purpose: The goal of this study is to compare endothelial small RNA transcriptome to identify the target of OASL under basal or stimulated conditions by utilizing miRNA-seq. Methods: Endothelial miRNA profilies of siCTL or siOASL transfected HUVECs were generated by illumina sequencing method, in duplicate. After sequencing, the raw sequence reads are filtered based on quality. The adapter sequences are also trimmed off the raw sequence reads. rRNA removed reads are sequentially aligned to reference genome (GRCh38) and miRNA prediction is performed by miRDeep2. Results: We identified known miRNA in species (miRDeep2) in the HUVECs transfected with siCTL or siOASL. The expression profile of mature miRNA is used to analyze differentially expressed miRNA(DE miRNA). Conclusions: Our study represents the first analysis of endothelial miRNA profiles affected by OASL knockdown with biologic replicates.
Project description:A cDNA library was constructed by Novogene (CA, USA) using a Small RNA Sample Pre Kit, and Illumina sequencing was conducted according to company workflow, using 20 million reads. Raw data were filtered for quality as determined by reads with a quality score > 5, reads containing N < 10%, no 5' primer contaminants, and reads with a 3' primer and insert tag. The 3' primer sequence was trimmed and reads with a poly A/T/G/C were removed
Project description:The goals of this study are to use Next-generation sequencing (NGS)to detect bacterial mRNA profiles of E.coli DH5a in response to 0, 20ug/L and 2 mg/L triclosan for 2 h, in triplicate, using Illumina HiSeq 2500.The NGS QC toolkit (version 2.3.3) was used to treat the raw sequence reads to trim the 3’-end residual adaptors and primers, and the ambiguous characters in the reads were removed. Then, the sequence reads consisting of at least 85% bases were progressively trimmed at the 3’-ends until a quality value ≥ 20 were kept. Downstream analyses were performed using the generated clean reads of no shorter than 75 bp. The clean reads of each sample were aligned to the E. coli reference genome (NC_000913) using SeqAlto (version 0.5). Cufflinks (version 2.2.1) was used to calculate the strand-specific coverage for each gene, and to analyze the differential expression in triplicate bacterial cell cultures. The statistical analyses and visualization were conducted using CummeRbund package in R (http://compbio.mit.edu/cummeRbund/). Gene expression was calculated as fragments per kilobase of a gene per million mapped reads (FPKM, a normalized value generated from the frequency of detection and the length of a given gene.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. Methods: spleen mRNA profiles of 8-weeks-old WT and lncRNA-ISIR, used to be called lnRNA-IRF3, knockout mice were generated by deep sequencing, using BGISEQ-500 system. The sequence reads that passed quality filters were analyzed at the transcriptional level with RSEM (RNA-seq by Expectation Maximization). Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.