Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA383809 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Purpose: The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) of U251 cells with endogenous Gcn5 depletion and reconstituted expression of WT Flag-rGcn5 or Flag-rGcn5 Y645A / endogenous DLST depletion and reconstituted expression of WT V5-rDLST or V5-rDLST R224A/K226E Transcriptomes. And quantitatively analyze the cell signaling pathways that regulated by the studied mutants. Methods: total mRNA was extracted from U251 cells with endogenous Gcn5 depletion and reconstituted expression of WT Flag-rGcn5 or Flag-rGcn5 Y645A / endogenous DLST depletion and reconstituted expression of WT V5-rDLST or V5-rDLST R224A/K226E.llumina compatible libraries were prepared by using the KAPA Stranded mRNA-Seq Library Preparation Kit for Illumina® platforms (KAPA Biosystems). In brief, 250 ng of total RNA was enriched for poly-A-tailed mRNA by using Kapa’s mRNA Capture beads. Poly-A-enriched RNA was fragmented to a median size of 150 bp by using chemical fragmentation and converted into double-stranded cDNA with dUTP incorporated into the second cDNA strand. The ends of the double-stranded cDNA were polished, 5’-phosphorylated, and 3’-A-tailed for the ligation of indexed adapters. Adapter-ligated DNA fragments were amplified by 8 cycles of PCR. The strand with incorporated dUTP was not amplified. The resulting libraries were quantified by qPCR and assessed for size distribution by using the 4200 TapeStation (Agilent Technologies), and then multiplexed, 4 per pool, and sequenced on the Illumina’s NextSeq500 by using the mid-output, 75-bp paired-end read format. Results: Using an optimized data analysis workflow, we mapped about 50 million sequence reads per sample to the human genome and quantified the expression of protein coding genes in the U251 cells of WT Gcn5 and Gcn5 Y645A mutant/WT DLST and DLST R224A/K226E mutant. Altered expression of 3 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Conclusions: Our study represents the first detailed analysis of U251 cells with endogenous Gcn5 depletion and reconstituted expression of WT Flag-rGcn5 or Flag-rGcn5 Y645A / endogenous DLST depletion and reconstituted expression of WT V5-rDLST or V5-rDLST R224A/K226E Transcriptomes. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that Gcn5 Y45A mutant and DLST R224A/K226E mutant, which reduce histone H3 K79 succinylation, can regulate several cell signaling pathways that important for cancer cell proliferation.
2017-12-13 | GSE98050 | GEO
Project description:Compound heterozygous or homozygous GBA1 mutations lead to Gaucher disease. Furthermore, GBA1 mutations are the most frequent risk factor for Parkinson’s disease. To get a better understanding of the pathological mechanisms, we generated inducible V5-Flag-Tag, V5-Flag-tagged WT, E326K and L444P mutant Flp-In™T-REx™-HEK293 and performed interatomic analysis.
2023-05-10 | PXD032155 | Pride
Project description:Carboxy-terminally tagged MOZ (Flag-V5-BIO tagged) was detected by ChIP-seq using anti-V5 antibody (Sigma, A7345) to precipitate chromatin associated with MOZ
2019-07-23 | GSE134607 | GEO
Project description:to find functional relevance between SMC5/6 complex and SAGA histone acetyltransferase complex in Schizosaccharomyces pombe by chromatin immunoprecipitation of Nse4-FLAG tagged protein from gcn5, ubp8 and ada2 deletion strains and 2 control strains (WT - Nse4-FLAG, 501 - no tag).
2023-02-20 | E-MTAB-12401 | biostudies-arrayexpress
Project description:We report the nuclear Gcn5/α-KGDH complex plays vital role in histone H3 K79 succinylation. By obtaining over 731 million reads from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of histone H3 K79 succinylation, Gcn5, and α-KGDH in U251 cells.
2017-12-13 | GSE97994 | GEO
Project description:Here we report the use of RPL22-Flag, RPL22-V5, and RPL22-HA to co-immunoprecipitate RNA from specific cell populations
2020-05-01 | GSE149076 | GEO
Project description:Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is the method of choice to study function of transcription factors in cell fate determination. This technique faces two major obstacles when applied to developmental studies: the availability of ChIP grade antibodies and access to sufficient quantities of the cells of interest. We present a robust method for the genome-wide analysis of transcription factor binding during development in highly homogenous cells in defined developmental states. It combines efficient embryonic stem cell (ESC) differentiation protocols with an inducible system of tagged transcription factors to enable affinity based assays such as ChIP-seq during lineage specific development. To validate the system, we compared the activity and genomic binding of native and V5-tagged Olig2 in motor neuron progenitors and Flag-tagged and V5-tagged Hoxc9 in motor neurons. We find that tagging transcription factors and expressing them alongside their endogenous counterparts does not alter their function or genomic association. The technology presented here can be applied to known as well as novel DNA-binding proteins. In combination with a suitable ESC differentiation paradigm it can be applied to determine how lineage-specific transcriptional networks are established and regulated. The aim of this study is to validate a platform for analysis of transcription factor binding that combines directed differentiation of ES cells with an inducible system of tagged transcription factors. Here, we perform ChIP-seq analysis to compare binding profiles of Olig2 as found using a native antibody and anti-V5 against the epitope tag. We also compare the ChIP-seq profiles of Hoxc9 as found using two independent epitope tags (V5 and FLAG). In all, there are 6 Illumina sequence datasets in this submission, including one replicate for each of native Olig2, iOlig2-V5 and FLAG-iHoxc9, two replicates of iHoxc9-V5, and a pseudo-ChIP control using anti-V5 in a non-induced iOlig2 cell-line. The differentiation of ventral motor neurons is induced by treating embryonic stem cell cultures with retinoic acid and hedgehog.
2011-11-12 | E-GEOD-30882 | biostudies-arrayexpress
Project description:Circulating microRNAs (miRNA) are relatively stable in plasma and are a new class of disease biomarkers. Here we present evidence that human high-density lipoprotein (HDL) transports endogenous miRNAs and delivers them to recipient cells with functional targeting capabilities. Highly-purified fractions of human HDL contain small RNAs, and the HDL-miRNA profile from normal subjects is significantly different than familial hypercholesterolemia subjects. miRNAs were demonstrated to associate with both native and reconstituted HDL particles, and reconstituted HDL injected into mice retrieved distinct miRNA profiles from normal and atherogenic models. Cellular export of miRNAs to HDL was demonstrated to be regulated by neutral sphingomyelinase. HDL-mediated delivery of miRNAs to recipient cells was demonstrated to be scavenger receptor BI-dependent. Furthermore, HDL delivery of both exogenous and endogenous miRNAs resulted in the direct targeting of mRNA reporters. Notably, HDL-miRNA from atherosclerotic subjects induced differential gene expression, with significant loss of conserved mRNA targets in cultured hepatocytes. Collectively, these observations suggest that HDL participates in a novel mechanism of intercellular communication involving the transport and delivery of miRNAs. Mouse HDL signatures from WT and LDLR-/- high fat diet Profiled HDL miRNA Signatures from N=4 WT; n=5 LDLR-/- HF mice
2011-03-23 | E-GEOD-25150 | biostudies-arrayexpress
Project description:Circulating microRNAs (miRNA) are relatively stable in plasma and are a new class of disease biomarkers. Here we present evidence that human high-density lipoprotein (HDL) transports endogenous miRNAs and delivers them to recipient cells with functional targeting capabilities. Highly-purified fractions of human HDL contain small RNAs, and the HDL-miRNA profile from normal subjects is significantly different than familial hypercholesterolemia subjects. miRNAs were demonstrated to associate with both native and reconstituted HDL particles, and reconstituted HDL injected into mice retrieved distinct miRNA profiles from normal and atherogenic models. Cellular export of miRNAs to HDL was demonstrated to be regulated by neutral sphingomyelinase. HDL-mediated delivery of miRNAs to recipient cells was demonstrated to be scavenger receptor BI-dependent. Furthermore, HDL delivery of both exogenous and endogenous miRNAs resulted in the direct targeting of mRNA reporters. Notably, HDL-miRNA from atherosclerotic subjects induced differential gene expression, with significant loss of conserved mRNA targets in cultured hepatocytes. Collectively, these observations suggest that HDL participates in a novel mechanism of intercellular communication involving the transport and delivery of miRNAs. microRNA signatures retrieved from rHDL injected into WT, ApoE-/- chow, ApoE-/-high fat diet Profiled rHDL miRNA signatures from n=3 WT; n=3 ApoE-/- chow, n=4 ApoE-/- high fat mice
2011-03-23 | E-GEOD-25424 | biostudies-arrayexpress
Project description:af31_haf2_gcn5 - comparison ws vs haf2_gcn5 - Genes whose expression during seed germination is affected by the mutation of two histone acetyltransferase genes GCN5 and HAF2 (the later encodes TAF1). - mRNA extraxted from 72h germinating seed of the mutants gcn5 and haf2 were used for comparison with Ws seed at the same satge. 2 repetitions techniques + 1 repetition biologique. Keywords: wt vs mutant comparison
2007-06-20 | GSE7977 | GEO