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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-Seq of head tissue from Drosophila melanogaster Wild Type and Adar5G1dAdar-/- mutant

ABSTRACT: Purpose: Validation of Drosophila A-to-I editing sites Methods: We collected heads of 5 day old male dAdar-/- mutant (y, Adar5G1, w)26 and wild type (w1118) flies. Poly(A)+ RNA was used to prepare RNA-seq libraries which were subsequently sequenced single-end by an Illumina GAII Results:We builded a framework to identify RNA editing events using RNA-seq data alone in Drosophila. To validate whether the identified A-to-G sites were bona fide A-to-I editing events, we performed RNA-seq for the D.melanogaster wild-type strain (w1118) and for the Adar5G1 null mutant that eliminates RNA editing. We found that our method achieved high accuracy; 98.2% of all A-to-G sites showed only adenosine in the Adar5G1 sample Conclusions: We anticipate that our method will be very effective in the future to identify RNA editing events in different species. mRNA profiles of heads of 5 day old male dAdar-/- mutant (y, Adar5G1, w)26 and wild type (w1118) flies

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Rui Zhang

PROVIDER: E-GEOD-42815 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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RNA-Seq of head tissue from Drosophila melanogaster Wild Type and Adar5G1dAdar-/- mutant

Project description:Purpose: Validation of Drosophila A-to-I editing sites Methods: We collected heads of 5 day old male dAdar-/- mutant (y, Adar5G1, w)26 and wild type (w1118) flies. Poly(A)+ RNA was used to prepare RNA-seq libraries which were subsequently sequenced single-end by an Illumina GAII Results:We builded a framework to identify RNA editing events using RNA-seq data alone in Drosophila. To validate whether the identified A-to-G sites were bona fide A-to-I editing events, we performed RNA-seq for the D.melanogaster wild-type strain (w1118) and for the Adar5G1 null mutant that eliminates RNA editing. We found that our method achieved high accuracy; 98.2% of all A-to-G sites showed only adenosine in the Adar5G1 sample Conclusions: We anticipate that our method will be very effective in the future to identify RNA editing events in different species.

2012-12-20 | GSE42815 | GEO

Genome-wide identification and functional analysis of Apobec-1 mediated C-to-U RNA editing in mouse small intestine and liver

Project description:The genome-wide identification, tissue-specificity and functional implications of Apobec-1 mediated C-to-U RNA editing remains incomplete. Deep sequencing, data filtering and validation from wild-type and Apobec-1 deficient mice revealed 56 novel editing sites in 54 intestinal mRNAs and 22 novel sites in 17 liver mRNAs (74-81% true-positive), all within 3' untranslated regions. Eleven of 17 liver RNAs shared editing sites with intestinal RNAs, while 6 sites were unique to liver. Changes in RNA editing led to corresponding changes in intestinal mRNA and protein levels in 11 genes. We found distinctive polysome profiles for several editing targets and demonstrated nuclear but not cytoplasmic editing of novel exonic sites in intestinal (but not hepatic) apoB RNA. RNA editing was validated using cell-free extracts from wild-type but not Apobec-1 deficient mice. These studies define selective, tissue-specific targets of Apobec-1 dependent RNA editing and show the functional consequences of editing are both transcript- and tissue-specific. Examination of C-to-U RNA editing in mouse liver and intestine

2014-05-23 | E-GEOD-57910 | biostudies-arrayexpress

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR) [RNA-seq]

Project description:Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery. HepG2 and K562 cell lines were stably transfected with plasmids containing siRNA designed to specifically knock down ADAR expression (ADAR KD). This in order to examine how ADAR affects alternative splicing globally.

2013-06-17 | E-GEOD-47997 | biostudies-arrayexpress

Phosphoproteomic screening identifies physiological substrates of dTSSK kinase

Project description:TO identified its potential phosphorylated substrates by investigating differential phosphorylated sites in the testes of wild-type (w1118) and dTSSK-/- male flies using quantitative phosphoproteomic analysis

2023-07-20 | PXD038767 | Pride

Transcription profiling of Drosophila heads heads from adult males, w118 strain

Project description:The w1118 strain is an inbred lab stock widely used in genetic and transgenic studies. w1118 males have a median life span of 35 days. Adult males were collected within 24 hr after eclosion. Approximately 200 flies were maintained in constant darkness in each food bottle at 25Â°C and 70% humidity and were transferred to fresh bottles every 3-4 days. Transcripts were harvested at d3 and d47. To separate the head from the rest of the body, flies were frozen and briefly vortexed in liquid nitrogen. Fly heads were collected using a sieve which retained fly bodies. Total RNA was extracted using Trizol (GIBCO/BRL). Poly(A) RNA was isolated using Oligotex resin (Qiagen). Samples were profiled with Affymetrix DrosGenome1 GeneChips, using standard Affymetrix protocol.

2008-06-16 | E-GEOD-826 | biostudies-arrayexpress

Evaluating the Impact of Sequencing Depth on Transcriptome Profiling in Human Adipose

Project description:Recent advances in RNA sequencing (RNA-Seq) have enabled the discovery of novel transcriptomic variations that are not possible with traditional microarray-based methods. Tissue and cell specific transcriptome changes during pathophysiological stress, in disease cases versus controls and in response to therapies are of particular interest to investigators studying cardiometabolic diseases. Thus, knowledge on the relationships between sequencing depth and detection of transcriptomic variation is needed for designing RNA-Seq experiments and for interpreting results of analyses. Using deeply sequenced RNA-Seq data derived from adipose of a healthy individual before and after systemic administration of endotoxin (LPS), we investigated the sequencing depths needed for studies of gene expression and alternative splicing (AS). We found that to detect expressed genes and AS events, ~100 million (M) filtered reads were needed. However, the requirement on sequencing depth for the detection of LPS modulated differential expression (DE) and differential alternative splicing (DAS) was much higher. To detect 80% of events, ~300M filtered reads were needed for DE analysis whereas at least 400M filtered reads were necessary for detecting DAS. Although the majority of expressed genes and AS events can be detected with modest sequencing depths (~100M filtered reads), the estimated gene expression levels and exon/intron inclusion levels were less accurate. We report the first study that evaluates the relationship between RNA-Seq depth and the ability to detect DE and DAS in human adipose. Our results suggest that a much higher sequencing depth is needed to reliably identify DAS events than for DE genes. Random sampling the RNA-seq data in different depth for gene and alternative-splicing analysis

2013-07-12 | E-GEOD-46323 | biostudies-arrayexpress

Nascent-seq indicates widespread cotranscriptional RNA editing in Drosophila

2012-05-31 | GSE37232 | GEO

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

Aging in heads from adult Drosophila males, w118 strain

Project description:The w1118 strain is an inbred lab stock widely used in genetic and transgenic studies. w1118 males have a median life span of 35 days. Adult males were collected within 24 hr after eclosion. Approximately 200 flies were maintained in constant darkness in each food bottle at 25°C and 70% humidity and were transferred to fresh bottles every 3-4 days. Transcripts were harvested at d3 and d47. To separate the head from the rest of the body, flies were frozen and briefly vortexed in liquid nitrogen. Fly heads were collected using a sieve which retained fly bodies. Total RNA was extracted using Trizol (GIBCO/BRL). Poly(A) RNA was isolated using Oligotex resin (Qiagen). Samples were profiled with Affymetrix DrosGenome1 GeneChips, using standard Affymetrix protocol. Keywords: time-course

2004-01-18 | GSE826 | GEO

RNA-seq of wild type and mutant Drosophila flies with a deletion of the gene CG16717 (dMPPED)

Project description:Flies deficient in the gene CG16716 were created and showed a reduced life span when compared to wild type w1118 flies. Flies were harvested on day 35 (both wild type and mutant flies), RNA prepared and subjected to RNA-seq.

2021-03-31 | E-MTAB-9081 | biostudies-arrayexpress

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