Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Sdhaf4-/- Cardiac Transcriptomes
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE163809 | GEO | 2022/03/24
REPOSITORIES: GEO
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Project description:Summary Purpose: By means of high-throughput data analysis (high-throughput sequencing or next-generation sequencing -NGS-), we addressed the effect of the absence of Ikaros and the Notch pathway activation in mouse fetal liver erythroid cells. Specifically, the goals of this study are (i) to characterize the transcriptome of erythroid cells in the absence of Ikaros, when the Notch pathway is activated or not (by RNA-seq); (ii) to identify patterns in the modification of gene regulation according to the variable conditions; and (iii) to obtain information on mechanisms involved in the variation of gene regulation. Methods: Erythroid cells obtained from Ikaros wild type (WT) or Ikaros knockout (Null) mouse fetal livers at the embryonic stage e14.5. These cells were co-cultured for 48 h on OP9 or OP9-DL1 cells. mRNA profiles were generated by NGS, in biological triplicate, using Illumina cBot 2 System. The quality of the raw reads was assessed with FASTQC. Report show no pool imbalance. After examining the quality of the raw reads, no trimming was deemed necessary. The reads were aligned to the GRCm38/mm10 genome with TopHat. The raw alignment counts were calculated with htseq-count. DESeq2 calculates the differential expression of genes directly from the raw alignment counts calculated with htseq-count. The output from DESeq2 includes the raw counts normalized relative to the total number of reads. The log2 fold change is an estimate of the fold change between the conditions, based on the distribution of the reads. qRT-PCR validation was performed using specific primer sets in real-time PCR with SYBR Green. Results. Based on expression profiles, all samples clustered correctly. The differential expression analysis was perform with all samples. For subsequent analysis, 2452 differentially expressed genes were identified in Ikaros WT vs Ikaros null cells cultured on OP9 cells, using a log2 ≥ 1 and a p value <0.005. Additionally, 2847 differentially expressed genes were identified in Ikaros WT vs Ikaros Null cells cultured on OP9-DL1 cells, using a log2 ≥ 1 and a p value <0.005. Among genes activated by Notch (cells cultured on OP9-DL1), 616 genes were repressed in the absence of Ikaros (Ikaros Null vs WT cells; log2≥ -0.8 and a p value <0.005) and 1558 genes were overexpressed in in the absence of Ikaros (Ikaros Null vs WT cells: log2≥ 0.8 and a p value <0.005), using adjusted p value <0.005. Modified expression or immunoprecipitation of several gene transcripts was confirmed with qRT-PCR. Conclusions (Summary): The results obtained demonstrate that Ikaros can favour repression of Notch target genes but can also be required for proper activation on Notch targets upon Notch pathway activation.
2020-12-18 | GSE122951 | GEO
Project description:Purpose: The goals of this study are to confirm whether CD5-2 works through VE-cadherin Methods: mRNA profiles of HUVEC and VE-cadherin-/- EC treated with either control or CD5-2 were generated by deep sequencing using Illumina Hiseq4000 platform and 150 bp paired-end reads were generated. Reference genome and gene model annotation files were downloaded from genome website browser (NCBI/UCSC/Ensembl) directly. Indices of the reference genome were built using Bowtie (v2.2.3) and paired-end clean reads were aligned to the reference genome using TopHat (v2.0.12). Differentially expressed genes were then determined using DESeq R package (v1.12.0). The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the False Discovery Rate (FDR). Genes with an adjusted P-value<0.05 and FDR<0.005 were assigned as differentially expressed. Results: 585 differentially expressed genes (DEGs) in CD5-2 treated ECs compared with control treated ECs (P < 0.05; false discovery rate [FDR] ≤ 0.005). In stark contrast to that seen in normal ECs, in VE-cadherin-/- ECs, no gene was significantly differently expressed between control and CD5-2 treated group when using the same cut-off criteria, Conclusions: Our study suggests VE-cadherin is essential for the CD5-2 mediated gene changes
2020-05-07 | GSE149948 | GEO
Project description:In this study, we analyzed the impact of a mutation in the wrn-1 gene compared to wild type worms and the dietary supplementation of vitamin C on the global mRNA expression of the whole C. elegans by the RNA-seq technology. Whole C. elegans mRNA profiles at the L4 stage of wild type and wrn-1(gk99) mutant animals treated with or without 10 mM ascorbate were generated by deep sequencing, in triplicate, using the HiSeq 2000 machine form Illumina. Detailed statistics on the quality of the reads were calculated with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The 50 base pairs raw sequences were aligned on the C. elegans ce10/W220 genome with TopHat using the Ensembl annotations provided with the Illumina iGenomes. The htseq-count software (http://www-huber.embl.de/users/anders/HTSeq) was used to count the number of reads aligned to each gene. These counts were then normalized relative to the sequencing depth with DESeq.
2014-10-07 | E-GEOD-54173 | biostudies-arrayexpress
Project description:Transcriptomic profile of human adipose tissue progenitor cells was performed as follows. For AmpliSeq transcriptome sequencing library construction, AmpliSeq™ Library PLUS, AmpliSeq Transcriptome Human Gene Expression Panel and AmpliSeq CD indexes SetA kits were purchased from Illumina and sequencing libraries were constructed as described in AmpliSeq for Illumina Transcriptome Human Gene Expression Panel reference guide (Illumina). Equimolar concentrations of libraries were pooled at 4 nM and denatured and diluted as described in Denature and Dilute Libraries Guide (Illumina) and adjusted to final concentration of 1.4 pM. Resulting library was sequenced on NextSeq 500 using NextSeq 500/550 High Output v2 kit with 2 X 151 bp cycle. Generated raw files were converted to FASTQ files and used for data analysis. AmpliSeq transcriptome FASTQ files were analyzed on Array studio V10.0 (Omicsoft, Qiagen). Following raw read QC, first and last 10 bases were trimmed and mapped to reference genome Human.B38. The read count data was generated using GeneModel RefGene20170606. Resulting data was normalized by DESeq package, transformed to log2 value and used for ANOVA analyses.
2023-01-12 | GSE222749 | GEO
Project description:The purpose of this study is to identify differentially expressed transcription factor genes during early plant growth. A total amount of 3 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. We used an optimized data analysis workflow, and mapped about 45 million sequence clean reads per sample to the Arabidopsis genome and identified 33,685 transcripts in 2 and 6 dpg (day post germination) old wild type plant (Col-0). Among them, there are 9321 genes differentially expresses from 2- and 6-d-old plants, and 479 differentially expresses transcription factor (TF) genes. Of these genes, 344 TF genes up regulated and 135 down regulated in 6-d-old plants compared with 2-d-old plants, with a fold change ≥2, ≤0.6 and p value <0.05. The P values were adjusted using the Benjamini & Hochberg method. Corrected P-value of 0.005 and log2(Fold change) of 1 were set as the threshold for significantly differential expression. This study will facilitate analysis related with developmental study in early plant growth stage.
2018-12-31 | GSE119325 | GEO
Project description:We report transcriptional profiles of aortic valve tissue from calcific aortic valve disease (CAVD) and normal control (non-CAVD). We collected the aortic valve tissues from five patients with CAVD who underwent aortic valve replacement due to severe aortic valve stenosis. Aortic valve samples from patients with non-calcified aortic valve resection due to heart transplantation (recipient heart) or aortic dissection were collected as the control (non-CAVD). The inclusion criteria for CAVD group were as follows: 50-75 years old; undergoing aortic valve replacement due to severe AVS with significantly valvular calcification. The inclusion criteria for non-CAVD group were as follows: non-calcified aortic valve resection due to heart transplantation (recipient heart) or aortic dissection. For each sample, total RNA was extracted, a cDNA library was generated, and an Illumina NovaSeq 6000 was used to sequence each sample. Stringtie software was used to count the fragment within each gene, and TMM algorithm was used for normalization. Differential expression analysis was performed using R package edgeR. Differentially expressed RNAs with |log2(FC)| value >1, q value [false discovery rate (FDR) adjusted P-value] <0.05, and one group’s mean fragments per kilobase of exon per million reads mapped (FPKM) >1, were assigned as differentially-expressed genes (DEGs).
2023-06-28 | GSE235995 | GEO
Project description:To explore the functions for Phb2 gene in early retinal degeneration, we performed RNA-seq analysis on the bulk retinas from control and photoreceptor-specific knockout Phb2 mice at 6 weeks of age. Compared with control mice, there were 778 differentially expressed genes (log2 |fold change|> 1, log10 adjusted p-values < 0.05) identified to be associated with distinct biological processes. 585 genes were significantly upregulated and 192 genes were significantly downregulated in photoreceptor cell-specific knockout Phb2 mice. The KEGG pathways analysis of up-regulated and down-regulated genes and ranking the enrichment pathways according to the corrected p-value.To explore the critical signaling pathways that might be affected by knockout Phb2 in retinal photoreceptors, we performed RNA-seq analysis on 6-week-old C57BL/6J photoreceptor cell-specific knockout Phb2 mice and control mice. Compared with control mice, there were 778 differentially expressed genes (log2 |fold change|> 1, log10 adjusted p-values < 0.05) identified to be associated with distinct biological processes. 585 genes were significantly upregulated and 192 genes were significantly downregulated in photoreceptor cell-specific knockout Phb2 mice. The KEGG pathways analysis of up-regulated and down-regulated genes and ranking the enrichment pathways according to the corrected p-value.
2024-12-31 | GSE246802 | GEO
Project description:Pyruvate kinase M2 (PKM2), as the terminal and last rate-limiting enzyme of the glycolytic pathway, is an ideal enzyme for regulating metabolic phenotype. PKM2 tetramer activation has shown a protective role against diabetic kidney disease (DKD). However, the molecular mechanisms involved in diabetic tubular has not been investigated so far. In this study, we performed transcriptome gene expression profiling in human renal proximal tubular epithelial cell line (HK-2 cells) treated with high D-glucose (HG) for 7 days before the addition of 10-μM TEPP-46, an activator of PKM2 tetramerization, for a further 1 day in the presence of HG. Afterwards, we analyzed the differentially expressed (DE) genes and investigated gene relationships based on weighted gene co-expression network analysis (WGCNA). The results showed that 2,902 DE genes were identified (adjusted P-value ≤ 0.05), where 2,509 DE genes (86.46%) were co-expressed in the key module. Four extremely down-regulated DE genes (HSPA8, HSPA2, HSPA1B and ARRB1) and three extremely up-regulated DE genes (GADD45A, IGFBP3 and SIAH1) enriched in the down-regulated endocytosis (hsa04144) and up-regulated p53 signaling pathway (hsa04115), respectively, were validated by the qRT-PCR experiments. The qRT-PCR results showed that the relative expression levels of HSPA8 (adjusted P-value = 4.45×10-34 & log2(FC) = -1.12), HSPA2 (adjusted P-value = 6.09×10-14 & log2(FC) = -1.27), HSPA1B (adjusted P-value = 1.14×10-11 & log2(FC) = -1.02) and ARRB1 (adjusted P-value = 2.60×10-5 & log2(FC) = -1.13) were significantly different (P-value < 0.05) from case group to control group. Furthermore, the interactions and predicted microRNAs of the key genes (HSPA8, HSPA2, HSPA1B and ARRB1) were visualized in networks. This study identified the key candidate transcriptomic biomarkers and biological pathways in the hyperglycemic HK-2 cells responding to the PKM2 activator TEPP-46. These results will be valuable for further research on PKM2 in DKD.
2022-08-29 | GSE205674 | GEO
Project description:Transcriptional analysis of the pancreatic islets of adult Paupar KO mice versus Paupar WT mice. Total RNA from Paupar WT and Paupar KO mice was converted into cDNA libraries (TruSeq RNA sample preparation kit, Illumina) according to manufacturer’s instructions. Sequencing was performed to a depth of 30 million, single-end reads in three biological replicates per genotype. Reads were aligned to the mouse genome (mm9) using HISAT2 v2.1.0 (Kim et al., 2013). Aligned reads were assigned to genes using annotations from Ensembl (Mus_musculus.GRCm38.73.gtf) and HTseq-count v0.10.0 (Anders et al., 2015). Differential expression across cohorts was assessed using DESeq2 v1.18 (Love, Huber, and Anders, 2014). Among the downregulated genes with a p-value < 0.05 genes important for alpha cell identity and function.
2019-10-10 | GSE121884 | GEO
Project description:Rapamycin-sensitive transgenic Arabidopsis lines (BP12) expressing yeast FK506 Binding Protein12 (FKBP12) were developed. Inhibition of TOR in BP12 plants by rapamycin resulted in slower overall root, leaf and shoot growth and development leading to poor nutrient uptake and light energy utilization. Genetic and physiological studies together with RNA-Seq and metabolite analysis of TOR-suppressed lines revealed that TOR regulates development and lifespan in Arabidopsis by restructuring cell growth, carbon and nitrogen metabolism, gene expression, ribosomal RNA and protein synthesis. Arabidopsis WT (Col)and BP12-2 (TOR knockdown line) seedlings at 15 DAG were treated with rapamycin for 3 days by transferring from 0.5 MS medium to 0.5 MS+10 ug/ml rapamycin. Triplicate samples of rapamycin treated WT and BP12-2 seedlings were used for RNA-Seq analysis (Illumina Hiseq 2000). Paired-end alignments were obtained through aligning short reads onto the reference Arabidopsis Genome (TAIR9) using Bowtie. More than 80% of the reads mapped onto the genome. Htseq-count was used to count the reads from the Bowtie derived output files. Differential expressed genes were identified using edgeR. The FDR-corrected P value for differential expression was set to be <=0.05.
2012-12-18 | E-GEOD-42968 | biostudies-arrayexpress