463e1bf6-c4db-433c-aff8-c35882bd9a52 - samples
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ABSTRACT:
PROVIDER: EGAD00001008512 | EGA |
REPOSITORIES: EGA
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Project description:To identify the expression profiles of circRNAs in peripheral blood mononuclear cells (PBMCs) from Ankylosing Spondylitis (AS) patients and healthy controls, we used circRNA sequencing to detect circRNA in 3 samples of PBMCs from AS patients and 3 samples of PBMCs from healthy controls. In brief, total RNA was extracted and purified using Magen Hipure Total RNA Mini Kit (Magen, Guangzhou, China), followed by constructing RNA sequencing libraries with KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems, Inc., MA, USA). After the construction of RNA sequencing libraries, Qubit 3.0 fluorometer (Invitrogen, CA, USA) and the Agilent 2100 bioanalyzer (Applied Biosystems, CA, USA) were used for quality control. Then, the PE150 mode of HiSeq X10 (Illumina Inc., CA, USA) was used for sequencing. Differentially expressed circRNAs were screened with criteria of fold-change>1.5 and p<0.05.
2022-01-05 | GSE178408 | GEO
Project description:Purpose: To investigate the function of PLIN2 in human uterine leiomyoma tumorigenesis, we generated genome-wide transcription profile of leiomyoma cells after PLIN2 knockdown. Methods: Primary leiomyoma cells were isolated from fresh uterine leiomyoma tissues and transfected with PLIN2 small interfering RNA (siRNA; 100 nM, #4392422) or non-targeting siRNA (100 nM, #4390844; Ambion, Foster City, CA) for 72 hours. Total RNA was isolated using the RNeasy Mini kit and RNA-seq library was completed per the manufacturer’s instructions using the KAPA Stranded RNA-seq Kit with RiboErase (#KK8483; KAPA Biosystems, Wilmington, MA). cDNA libraries were sequenced as single-end, 75 base-length reads on an Illumina NextSeq 500 instrument (Illumina, San Diego, CA) with an average read count of 27 million reads per sample. The quality of DNA reads, in fastq format, was evaluated using FastQC. Adapters were trimmed, and reads of poor quality or aligning to rRNA sequences were filtered. The cleaned reads were aligned to the Homo sapiens genome (hg19) using STAR. Read counts for each gene were calculated using htseq-count in conjunction with a gene annotation file for hg19 obtained from University of California Santa Cruz (http://genome.ucsc.edu). Results: A total of 3877 genes were found to be differentially expressed, with 2557 genes found to be upregulated and 1320 genes found to be downregulated by PLIN2 knockdown. Gene Ontology analysis (DAVID, https://david.ncifcrf.gov) identified cellular component organization/biogenesis (P=1.4E-27) and metabolic processes (P=1.6E-10) as the top two biological processes of genes upregulated by PLIN2 depletion. Kyoto Encyclopedia of Genes and Genomes (KEGG; DAVID) pathway analysis identified enrichment of genes involved in the cell cycle, pyruvate metabolism, fatty acid metabolism and degradation, purine and pyrimidine metabolism, apoptosis, and extracellular matrix component organization after PLIN2 depletion. Conclusions: These findings provide support for a role of PLIN2 depletion in activating metabolic activity within leiomyoma cells.
2020-01-24 | GSE128898 | GEO
Project description:Purpose: The purpose of this study is to measure the changes in liver transcriptome in response to short-term fasting between 7 and 13 h where the rats were dosed with 2 ml/kg of saline vehicle at 0 h Methods: Total RNA was isolated from the liver, using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA) and the direct-zol RNA Mini Prep kit (Zymo Research, Irvine, CA). The isolated RNA samples were then submitted to the Vanderbilt University Medical Center VANTAGE Core (Nashville, TN) for RNA quality determination and sequencing. Total RNA quality was assessed using a 2100 Bioanalyzer (Agilent, Santa Clara, CA). At least 200 ng of DNase-treated total RNA with high RNA integrity was used to generate poly-A-enriched mRNA libraries, using KAPA Stranded mRNA sample kits with indexed adaptors (Roche, Indianapolis, IN). Library quality was assessed using the 2100 Bioanalyzer (Agilent), and libraries were quantitated using KAPA library Quantification kits (Roche). Pooled libraries were subjected to 75-bp paired-end sequencing according to the manufacturer’s protocol (Illumina HiSeq3000, San Diego, CA). Results: No genes were were found to be differentially expressed with a false discovery rate less than 0.1 Conclusions: There were no significant changes in liver gene expression between 7 and 13 h of fasting
2019-03-19 | GSE123987 | GEO
Project description:Purpose: The purpose of this study is to measure the changes in liver transcriptome in response to short-term fasting between 5 and 10 h where the rats were dosed with 6 ml/kg of polyethylene glycol vehicle at 0 h Methods: Total RNA was isolated from the liver, using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA) and the direct-zol RNA Mini Prep kit (Zymo Research, Irvine, CA). The isolated RNA samples were then submitted to the Vanderbilt University Medical Center VANTAGE Core (Nashville, TN) for RNA quality determination and sequencing. Total RNA quality was assessed using a 2100 Bioanalyzer (Agilent, Santa Clara, CA). At least 200 ng of DNase-treated total RNA with high RNA integrity was used to generate poly-A-enriched mRNA libraries, using KAPA Stranded mRNA sample kits with indexed adaptors (Roche, Indianapolis, IN). Library quality was assessed using the 2100 Bioanalyzer (Agilent), and libraries were quantitated using KAPA library Quantification kits (Roche). Pooled libraries were subjected to 75-bp single-end sequencing according to the manufacturer’s protocol (Illumina HiSeq3000, San Diego, CA). Results: No genes were were found to be differentially expressed with a false discovery rate less than 0.1 Conclusions: There were no significant changes in liver gene expression between 5 and 10 h of fasting
2019-03-19 | GSE123935 | GEO
Project description:Purpose: The purpose of this study is to measure the changes in liver transcriptome in response to short-term fasting between 5 and 10 h where the rats were dosed with 2 ml/kg of corn oil vehicle at 0 h Methods: Total RNA was isolated from the liver, using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA) and the direct-zol RNA Mini Prep kit (Zymo Research, Irvine, CA). The isolated RNA samples were then submitted to the Vanderbilt University Medical Center VANTAGE Core (Nashville, TN) for RNA quality determination and sequencing. Total RNA quality was assessed using a 2100 Bioanalyzer (Agilent, Santa Clara, CA). At least 200 ng of DNase-treated total RNA with high RNA integrity was used to generate poly-A-enriched mRNA libraries, using KAPA Stranded mRNA sample kits with indexed adaptors (Roche, Indianapolis, IN). Library quality was assessed using the 2100 Bioanalyzer (Agilent), and libraries were quantitated using KAPA library Quantification kits (Roche). Pooled libraries were subjected to 150-bp paired-end sequencing according to the manufacturer’s protocol (Illumina NovaSeq6000, San Diego, CA). Results: Conclusions: There were 100 genes that could be mapped to the rat metabolic network iRno, with significant changes in expression between 5 and 10 h of fasting in the top 10% by effect size parameter b (b > 0.6).
2019-03-19 | GSE124004 | GEO
Project description:Purpose: With the advent of human genome sequencing project, circRNAs attracted widespread attention in cancer research due to its stable ring structure. Our aim was to identify differentially expressed circRNAs in GC and explore their potential roles in GC diagnosis and treatment. Methods: Total RNA from tissues was isolated by Hipure Total RNA Mini Kit (Magen, Germany). Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, California) was used for RNA concentration measurement while Agilent 2100 Bioanalyzer (Applied Biosystems, Carlsbad, CA) for RNA integrity estimation. A RIN value over 7.0 was considered eligible. RNA-seq library was prepared with approximately 2μg of total RNA using KAPA RNA HyperPrep Kit with RiboErase (HMR) for Illumina® (Kapa Biosystems, Inc., Woburn, MA). Briefly, total RNA was incubated at 37 °C for 30 min with 10 units RNase R (Epicentre Technologies, Madison, USA) after removing ribosomal RNA. Next, the ribominus RNase R (+) RNAs was fragmented and then first strand and directional second strand synthesis were performed. Then the A tailing and adapter ligation were performed with the purified cDNA. Finally, the purified, adapter-ligated DNA was amplified. Each library was diluted to 10 nM and pooled equimolar prior to clustering. Paired-End (PE150) sequencing was performed on all samples. Results: A total of 25,303 circRNA targets, including 20,036 known circRNAs and 5,267 uncharacterized circRNAs, were detected and defined. Among them, there were 2,007 circRNAs with statistically significant differences in expression in GC tissues, in which fold changes>2.0 and P<0.05 were identified.
2019-06-16 | GSE131414 | GEO
Project description:Purpose: We investigated the effect of the T4 MotB protein on E. coli gene expression Method: BL21(DE3) E. coli containing either pNW129 or pNW129-MotB were grown to early log phase (OD600 ~ 0.3) then induced with 0.2% arabinose for 20 minutes. Cells were then harvested and total RNA was isolated. The cDNA library was prepared using a modified RNATagSeq workflow as previously described (Shishkin, A.A. et al. 2015 Nat Methods). Optimum fragmentation of the total RNA samples in this library was determined to be 3 min at 94C in FastAP buffer (Thermo Fischer Scientific). The cDNA Library was run on a Bioanalyzer using the Agilent High Sensitivity DNA Kit to evaluate the quality of the library. The concentration of the cDNA library was determined by qPCR using the KAPA Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA) and CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Sequencing was performed by the NIDDK Genomics Core facility using a MiSeq system with the single-end 50 bp Sequencing Kit (Illumina, San Diego, CA, USA). RNA-seq data was processed as previously described using E. coli B str. DE3 (NC_012971.2) as the reference genome. Differential expression between conditions was represented as a fold change, and genes with both a fold change ≥2 and adjusted p value ≤ 0.05 were considered significant. Results: RNA-seq data revealed that the expression of genes encoded in the cryptic prograge Lambda DE3 as well as an additional 29 of E. coli genes were significantly increased after motB expression. Conclusion: T4 MotB is a DNA binding protein that compacts host DNA and disrupts H-NS dependent repression.
2020-06-11 | GSE152170 | GEO
Project description:Our aim is to explore the effect of Hydroxy-carboxylic Acid Receptor 1 on cardiomyocytes. Neonatal rat cardiomyocytes (NRCMs) were isolated and cultured. Subsequently, NRCMs were treated with the agonist of Hydroxy-carboxylic Acid Receptor 1 (HCAR1), 3Cl-HBA at 40 μM for 48 h(HBA1,HBA2 and HBA3) or DMSO as control(C1,C3 and C3). RNA was extracted using the KAPA RiboErase RNA-Seq kit (Roche, Basel, Switzerland), and was analysed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) for quality control. The libraries were sequenced on an Illumina HiSeq X Ten platform. DESeq2 was used to analyse RNA-seq data. Neonatal rat cardiomyocytes (NRCMs) were isolated and cultured. Subsequently, NRCMs were treated with the agonist of Hydroxy-carboxylic Acid Receptor 1 (HCAR1), 3Cl-HBA (40 μM for 48 h) or DMSO as control. RNA was extracted using the KAPA RiboErase RNA-Seq kit (Roche, Basel, Switzerland), and was analysed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) for quality control. The libraries were sequenced on an Illumina HiSeq X Ten platform.
2024-02-14 | GSE224822 | GEO
Project description:Purpose: We investigated the effect of the T4 MotB protein on E. coli gene expression Method: E. coli BL21 (DE3) containing either pNW129 or pNW129-MotB were grown to early log phase (OD600 ~ 0.3) then induced with 0.2% arabinose for 20 minutes. T4 phage added to the culture at MOI10. Cells were then harvested at 0, 5, and 10 min time points and total RNA was isolated. The cDNA library was prepared using a modified RNATagSeq workflow as previously described (Shishkin, A.A. et al. 2015 Nat Methods). Optimum fragmentation of the total RNA samples in this library was determined to be 3 min at 94C in FastAP buffer (Thermo Fischer Scientific). The cDNA Library was run on a Bioanalyzer using the Agilent High Sensitivity DNA Kit to evaluate the quality of the library. The concentration of the cDNA library was determined by qPCR using the KAPA Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA) and CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Sequencing was performed by the NIDDK Genomics Core facility using a MiSeq system with the single-end 50 bp Sequencing Kit (Illumina, San Diego, CA, USA). RNA-seq data was processed as previously described using E. coli str. K-12 substr. MG1655 (NC_000913.3) as the reference genome. Differential expression between conditions was represented as a fold change, and genes with both a fold change ≥2 or ≤ 0.5 and adjusted p value ≤ 0.05 were considered significant. Results: RNA-seq data revealed that the expression of 542 E. coli genes were significantly changed after motB expression. The expression of 704 and 706 E. coli genes were changed T4 5 min and 10 min post infection after MotB expression, respectively. After 5 min T4 infection, 34 T4 genes were significantly changed. Conclusion: T4 MotB modifies the pool of host tRNAs, creating a better infection for T4
2022-06-14 | GSE205715 | GEO
Project description:Purpose: We investigated the effect of the T4 MotB protein on E. coli gene expression Method: TOP10F' E. coli containing either pNW129 or pNW129-MotB were grown to early log phase (OD600 ~ 0.3) then induced with 0.2% arabinose for 20 minutes. T4 phage added to the culture at MOI10. Cells were then harvested at 0, 5, and 10 min time points and total RNA was isolated. The cDNA library was prepared using a modified RNATagSeq workflow as previously described (Shishkin, A.A. et al. 2015 Nat Methods). Optimum fragmentation of the total RNA samples in this library was determined to be 3 min at 94C in FastAP buffer (Thermo Fischer Scientific). The cDNA Library was run on a Bioanalyzer using the Agilent High Sensitivity DNA Kit to evaluate the quality of the library. The concentration of the cDNA library was determined by qPCR using the KAPA Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA) and CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Sequencing was performed by the NIDDK Genomics Core facility using a MiSeq system with the single-end 50 bp Sequencing Kit (Illumina, San Diego, CA, USA). RNA-seq data was processed as previously described using E. coli str. K-12 substr. MG1655 (NC_000913.3) as the reference genome. Differential expression between conditions was represented as a fold change, and genes with both a fold change ≥2 or ≤ 0.5 and adjusted p value ≤ 0.05 were considered significant. Results: RNA-seq data revealed that the expression of 542 E. coli genes were significantly changed after motB expression. The expression of 704 and 706 E. coli genes were changed T4 5 min and 10 min post infection after MotB expression, respectively. After 5 min T4 infection, 34 T4 genes were significantly changed. Conclusion: T4 MotB is a DNA binding protein that compacts host DNA and disrupts H-NS dependent repression.
2021-04-21 | GSE172467 | GEO