Browse
Submit Data
Databases
API
Help

Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

83 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Nascent mRNA profiling of LPS-stimulated mouse macrophages

ABSTRACT: To identify transcriptionally regulated genes in primary mouse macrophages stimulated with LPS with high sensitivity, we isolated nascent RNA following metabolic labelling with 4-thiouridine during the last 35 min before cell harvest, as recently described (Dolken et al. 2008 RNA 14:1959-72). Microarray analyses of nascent RNA identified substantially more probe sets as up-regulated after 45 min of LPS stimulation than parallel analyses of total cellular RNA. In contrast, 4.5 h after stimulation, up-regulated genes in total and nascent RNA largely overlapped. This approach therefore allowed a much more sensitive detection of early changes in transcription, and the respective genes are likely to be direct targets of LPS-regulated transcription factors. Keywords: Effect of LPS stimulation; comparison of changes in expression of total versus nascent RNA; timecourse Two completely independent experiments were performed. Macrophages were stimulated with 100 ng/ml LPS for 45 or 270 minutes. Thiouridine pulsing was done in last 35 minutes before harvest. Total RNA was isolated. Nascent RNA was subsequently purified.

ORGANISM(S): Mus musculus

SUBMITTER: Lars DÃ¶lken

PROVIDER: E-GEOD-20674 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Json Xml

Shared Molecules

Only show the datasets with similarity scores above: 0.5

Threshold

0.5

Similar Datasets

Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution

Project description:RNA synthesis and decay rates determine the steady-state levels of cellular RNAs. Metabolic tagging of newly transcribed RNA by 4-thiouridine (4sU) can reveal the relative contributions of RNA synthesis and decay rates. The kinetics of RNA processing, however, so far remained unresolved. Here, we show that ultra-short 4sU-tagging not only provides snap-shot pictures of eukaryotic gene expression but, when combined with progressive 4sU-tagging and RNA-seq, reveals global RNA processing kinetics at nucleotide resolution. Using this method, we identified classes of rapidly and slowly spliced/degraded introns. Interestingly, each class of splicing kinetics was characterized by a distinct association with intron length, gene length and splice site strength. For a large group of introns, we also observed long lasting retention in the primary transcript, but efficient secondary splicing or degradation at later time points. Finally, we show that processing of most, but not all small nucleolar (sno)RNA-containing introns is remarkably inefficient with the majority of introns being spliced and degraded rather than processed into mature snoRNAs. In summary, our study yields unparalleled insights into the kinetics of RNA processing and provides the tools to study molecular mechanisms of RNA processing and their contribution to the regulation of gene expression. 4sU-tagging was performed in human DG75 B-cells by adding 500 uM 4sU to cell culture medium for 5, 10, 15, 20 or 60 min. Following isolation of total cellular RNA, this was separated into nascent and untagged, pre-existing RNA. Nascent RNA as well as total and untagged RNA from 60 min 4sU-tagging were subjected to SOLiD sequencing (SOLiD II) obtaining 35 nt reads.

2012-04-25 | E-GEOD-31653 | biostudies-arrayexpress

Effect of cellular and viral miRNAs on target RNA half-life in three human B-cell lines

Project description:Total, nascent and unlabeled RNA were prepared following 1h of labeling with 100 µM 4-thiouridine and 3 replicates analyzed by Affymetrix Gene ST 1.0 arrays Transcript half-lives were determined in DG75-eGFP, DG75-10/12 and BCBL-1 based on nascent/total RNA ratios; unlabeled RNA was analyzed in addition

2010-11-04 | E-GEOD-18882 | biostudies-arrayexpress

Dissecting primary (translation independent) from secondary (translation dependent) IFN-mediated differential gene expression

Project description:NIH-3T3 cells were pretreated for 15 min with either DMSO (mock) or cycloheximide followed by addition of either mock, 100 U/ml IFNalpha or 100 U/ml IFNgamma for 1h. During the last 30 min, 500 µM 4-thiouridine was added to cell culture medium. Total cellular RNA was isolated using Trizol reagent and nascent RNA was purified as described (Dölken et al. RNA 2008) . Three replicates of nascent RNA were analyzed by Affymetrix Mouse Gene ST 1.0 arrays Primary (translation independent) from secondary (translation dependent) IFN-mediated differential gene expression were studied in NIH-3T3 fibroblasts by studying studying differential gene expression in presence and absence of Cycloheximide (CHX). In addition, the effect of 75 min CHX during the last 30 min of treatment was studied in nascent RNA.

2013-07-04 | E-GEOD-30457 | biostudies-arrayexpress

Coordinate Regulation of Enhancer Transcription at Super-Enhancers

Project description:Using GRO-Seq, we find extensive regulation of enhancer RNAs (eRNA) within super-enhancers in response to lipopolysaccharide treatment in macrophages. Both activation and repression of gene expression are associated with super-enhancers and eRNA transcription dynamics. Co-treatment of LPS and the anti-inflammatory drug dexamethasone targeted specific super-enhancers by attenuating their eRNA expression, leading to reduced expression of key inflammatory genes. We propose that super-enhancers function as molecular rheostats integrating the binding profiles of key regulators to produce dynamic profiles of gene expression. Nascent transcriptome (GRO-Seq) analysis over a time course (0, 20, 60, 180 min) of Lipopolisaccharide and Dexamethasone signaling in mouse bone marrow-derived macrophages.

2014-12-25 | E-GEOD-60857 | biostudies-arrayexpress

Nascent mRNA profiling of LPS-stimulated mouse macrophages

Project description:To identify transcriptionally regulated genes in primary mouse macrophages stimulated with LPS with high sensitivity, we isolated nascent RNA following metabolic labelling with 4-thiouridine during the last 35 min before cell harvest, as recently described (Dolken et al. 2008 RNA 14:1959-72). Microarray analyses of nascent RNA identified substantially more probe sets as up-regulated after 45 min of LPS stimulation than parallel analyses of total cellular RNA. In contrast, 4.5 h after stimulation, up-regulated genes in total and nascent RNA largely overlapped. This approach therefore allowed a much more sensitive detection of early changes in transcription, and the respective genes are likely to be direct targets of LPS-regulated transcription factors. Keywords: Effect of LPS stimulation; comparison of changes in expression of total versus nascent RNA; timecourse

2010-04-25 | GSE20674 | GEO

The regulatory roles of Ezh2 in response to lipopolysaccharide (LPS) in macrophages and mice with conditional Ezh2 deletion with LysM-Cre system

Project description:The responses of macrophages to lipopolysaccharide (LPS) might determine the direction of clinical manifestations of sepsis, which is the immune response against severe infection. Meanwhile, the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, might interfere with LPS response. With a single LPS stimulation, Ezh2 null(Ezh2flox/flox; LysM-Crecre/−) macrophages demonstrated lower supernatant TNF-α than Ezh2 control (Ezh2fl/fl; LysM-Cre−/−), perhaps due to an upregulation of Socs3, which is a suppressor of cytokine signaling 3, due to the loss of the Ezh2 gene. In LPS tolerance, Ezh2 null macrophages indicated higher supernatant TNF-α and IL-6 than the control, supporting an impact of the loss of the Ezh2 inhibitory gene. In parallel, Ezh2 null mice demonstrated lower serum TNF-α and IL-6 than the control mice after an LPS injection, indicating a less severe LPS-induced hyper-inflammation in Ezh2 null mice. In conclusion, an absence of Ezh2 in macrophages resulted in less severe LPS-induced inflammation, as indicated by low serum cytokines, with less severe LPS tolerance, as demonstrated by higher cytokine production, partly through the upregulated Socs3.

2023-07-20 | PXD041265 | Pride

Long read sequencing of nascent RNA from S. cerevisiae and S. pombe

Project description:Long read SMRT cDNA sequencing of nascent RNA from exponentially growing S. cerevisiae and S. pombe cells was employed to obtain transcription elongation and splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010). The nascent 3â?? end was labeled with a 3â?? DNA adaptor through ligation. The adaptor sequence served as template for full-length reverse transcription and double-stranded cDNA was obtained in a PCR (gene-specific or transcriptome-wide). SMRT DNA sequencing libraries were prepared subsequently. Nascent RNA profiles for mainly intron-containing genes were generated with long-read SMRT cDNA sequencing.

2016-03-24 | E-GEOD-70906 | biostudies-arrayexpress

Transcriptional profiling in macrophages in stimulated and unstimulated conditions.

Project description:Transcriptomes from macrophages at three stages were examined: a) Non-stimulated, b)Stimulated by Interleukin 4, c)Stimulated by LPS and Interferon gamma. Four biological replicate of each experiment were performed.

2018-05-31 | E-MTAB-6416 | biostudies-arrayexpress

Nascent-seq indicates widespread cotranscriptional RNA editing in Drosophila

Project description:The RNA editing enzyme ADAR chemically modifies adenosine (A) to inosine (I), which is interpreted by the ribosome as a guanosine. Here we assess cotranscriptional A-to-I editing in Drosophila, by isolating nascent RNA from adult fly heads and subjecting samples to high-throughput sequencing. There are a large number of edited sites within nascent exons. Nascent RNA from an ADAR null mutant strain was also sequenced, indicating that almost all A-to-I events require ADAR. Moreover, mRNA editing levels correlate with editing levels within the cognate nascent RNA sequence, indicating that the extent of editing is set cotranscriptionally. Surprisingly, the nascent data also identify an excess of intronic over exonic editing sites. These intronic sites occur preferentially within introns that are poorly spliced cotranscriptionally, suggesting a link between editing and splicing. We conclude that ADAR-mediated editing is more widespread than previously indicated and largely occurs cotranscriptionally. GSM914095: Fly genomic DNA sequencing. Sequenced on the Illumina GA II. GSM914102-GSM914113: Fly head nascent RNA profiles over 6 time points of a 12hr light:dark cycle in duplicate; sequenced on the Illumina GA II. GSM914114-GSM914119: Fly head nascent RNA profiles of yw, FM7, ADAR0 males in duplicate; sequenced on the HiSeq2000. GSM915213-GSM915214: Fly head mRNA profiles over 2 time points of a 12hr light:dark cycle; sequenced on the Illumina GA II. GSM915215-GSM915220: Fly head mRNA profiles over 6 time points of a 12hr light:dark cycle; paired-end sequenced on the Illumina GA II. GSM915221-GSM91526: Fly head mRNA profiles over 6 time points of a 12hr light:dark cycle; sequenced on the Illumina GA II.

2012-05-31 | E-GEOD-37232 | biostudies-arrayexpress

Targeted paired-end sequencing of nascent RNA from S. cerevisiae

Project description:Targeted paired-end sequencing of cDNA from unfragmented nascent RNA from exponentially growing S. cerevisiae cells was employed to obtain Pol II transcription elongation and splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010) or enriched from total RNA with polyadenylated RNA depletion. The nascent 3â end was labeled with a 3â DNA adaptor through ligation. A PCR with a forward primer in the first exon of select intron-containing genes amplifies nascent transcripts of specific genes and ensures sequencing adaptor attachment for paired-end sequencing. With this approach co-transcriptional splicing progression with distance from the intron end could be analyzed for 87 genes. Note that the unmapped and mapped data also include genes that did not pass the read coverage requirements in SMIT analysis. Nascent RNA profiles for mainly intron-containing genes were generated with paired-end sequencing with Illumina HiSeq technology.

2016-03-24 | E-GEOD-70907 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data