Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Sdhaf4-/- Cardiac Transcriptomes
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE163809 | GEO | 2022/03/24
REPOSITORIES: GEO
Similar Datasets
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 goal of this study is to compare the differently expressed genes in the wild type and the KDEL-tailed cysteine protease AtCEP1 knockout (atcep1) Arabidopsis using RNA-sequencing (RNA-seq). Methods: Arabidopsis buds mRNA profiles of anther development stages 5-6, 7-9, and 10-11 of the wild type (WT) and atcep1 mutant were generated by deep sequencing via Illumina HiSeqTM 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with the following steps: Remove reads with adaptor sequences; Remove reads in which the percentage of unknown bases (N) is greater than 10%; Remove low quality reads, in which the percentage of the low quality base (base with quality value ⤠5) is greater than 50%. The clean reads were mapped to the Arabidopsis reference genome and reference genes using SOAP aligner/SOAP2. No more than 2 mismatches were allowed in the alignment. The gene expression level was calculated using RPKM (Reads Per Kb per Million reads). Differential expression analysis between the wild type and the atcep1 mutant was performed using the DEGseq R package (1.12.0) based on normalized read counts. A corrected P value of < 0.005 and |log2Ratio| > 1 were set as the threshold for significantly differential expression. Results: We identified 872 genes showing significant differential expression, and in the atcep1 mutant, the upregulated genes significantly outnumbered the downregulated genes at the three time points. The GO analysis of the differently expressed genes showed that the expression of genes participating in anther tapetal secretory structure formation, pollen wall development, and tapetal cell wall generation, clearly changed. Arabidopsis buds mRNA profiles of development stages 5-6, 7-9, and 10-11 of the wild type (WT) and atcep1 muant were generated by deep sequencing via Illumina HiSeqTM 2000.
2016-03-25 | E-GEOD-50956 | biostudies-arrayexpress
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:RNA extraction and microarray analysis total RNA from long-term-cultured breast cancer stem cells of TV-circRGPD6-treated group and si-circRGPD6 group. Microarray experiments were conducted according to the manufacturer's instructions. To select the differentially expressed genes, we used threshold values of ≥ 2 and ≤2-fold change and a Benjamini-Hochberg corrected p value of 0.05. The data was Log2 transformed and median centered by genes using the Adjust Data function of Cluster 3.0 software then further analyzed with hierarchical clustering with average linkage (genes which value more than 100 were evaluated).
2022-02-12 | E-MTAB-7557 | biostudies-arrayexpress
Project description:RNA extraction and microarray analysis total RNA from long-term-cultured breast cancer stem cells of TV-circRGPD6-treated group and si-circRGPD6 group.The miRNA profiling was performed using Agilent miRNA array. Microarray experiments were conducted according to the manufacturer's instructions. To select the differentially expressed genes, we used threshold values of ≥ 2 and ≤2-fold change and a Benjamini-Hochberg corrected p value of 0.05. The data was Log2 transformed and median centered by genes using the Adjust Data function of Cluster 3.0 software then further analyzed with hierarchical clustering with average linkage (genes which value more than 100 were evaluated).
2022-02-13 | E-MTAB-7556 | biostudies-arrayexpress
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 used complementing high-throughput sequencing technologies, including RNA-seq, DNase I-seq, and ChIP-seq, to examine the distribution of DNase I hypersensitive sites and H3K4me2 histone marks, to infer transcription factor binding sites, to identify key pathways in Gefinitib resistant and sensitive cell lines, PC9R and PC9 respectively, and to verify that some of up-regulated genes in PC9R can be found in Gefinitib resistant patients, which indicated that our study has valid clinical relevance. Total mRNA profiles of PC9 and PC9R cell lines were generated by deep sequencing, in duplicate, using Illumina Hiseq2000.The mapping results of RNA-seq reads by the TopHat algorithm (version 1.1.4) to the hg19 reference genome were used to count the number of reads per gene with the hgseq-count algorithm. Then, DESeq was used to detect the genes differentially expressed between the PC9 and PC9R samples, with a p-value of 0.05 as the threshold. DNase-Seq and ChIP-Seq raw reads were aligned with Bowtie0.12.9 to hg19. MACS2 (https://github.com/taoliu/MACS/downloads) was employed to call PC9-specific regions using PC9 cells as the treatment and PC9R cells as the control, with a q-value threshold (DNase-Seq: 10-2; ChIP-Seq: 10-3) to minimise false positives; then, the process was repeated with the treatment and control cell lines switched.
2017-11-20 | GSE63356 | 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
Project description:Purpose: The goal of this study is to compare the differently expressed genes in the wild type and the KDEL-tailed cysteine protease AtCEP1 knockout (atcep1) Arabidopsis using RNA-sequencing (RNA-seq). Methods: Arabidopsis buds mRNA profiles of anther development stages 5-6, 7-9, and 10-11 of the wild type (WT) and atcep1 mutant were generated by deep sequencing via Illumina HiSeqTM 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with the following steps: Remove reads with adaptor sequences; Remove reads in which the percentage of unknown bases (N) is greater than 10%; Remove low quality reads, in which the percentage of the low quality base (base with quality value ≤ 5) is greater than 50%. The clean reads were mapped to the Arabidopsis reference genome and reference genes using SOAP aligner/SOAP2. No more than 2 mismatches were allowed in the alignment. The gene expression level was calculated using RPKM (Reads Per Kb per Million reads). Differential expression analysis between the wild type and the atcep1 mutant was performed using the DEGseq R package (1.12.0) based on normalized read counts. A corrected P value of < 0.005 and |log2Ratio| > 1 were set as the threshold for significantly differential expression. Results: We identified 872 genes showing significant differential expression, and in the atcep1 mutant, the upregulated genes significantly outnumbered the downregulated genes at the three time points. The GO analysis of the differently expressed genes showed that the expression of genes participating in anther tapetal secretory structure formation, pollen wall development, and tapetal cell wall generation, clearly changed.
2016-03-25 | GSE50956 | GEO
Project description:Raw RNA-seq counts from reads aligned to the mouse genome (mm10 assembly) were generated with Htseq-count (PMID: 25260700). Differentially expressed genes between TPr and Ctl MSCs were calculated by DESeq2 (PMID: 25516281). Pre-ranked gene set enrichment was performed as recommended for RNA-seq data (PMID: 16199517). Custom R scripts were used to filter highly redundant biological processes. A false discovery rate of 0.05 was considered as an acceptable threshold for further investigation. All analyses were conducted in R (v3.6) or Python (v3.7) programming language. The ggplot2, ClusterProfiler and dplyr packages were used for data visualization. Student’s t-test was performed for normally distributed data to compute the p-value and the Benjamini-Hochberg procedure was used for adjusting the statistical inference of multiple comparisons.
2021-02-08 | GSE161773 | GEO