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Drosophila melanogaster
54 Downloadable Samples
Illumina HiSeq 2000
Description
We use mRNA-seq in combination with polysome profiling to determine translational status for all mRNAs in Drosophila mature oocytes and activated eggs. Puromycin-treated lysates are used as a negative control in polysome profiling experiments. Additionally, we use ribosome footprinting to globally measure translational efficiency of mRNAs in wild type mature oocytes as well as wild type and png mutant activated eggs. Overall design: Lysates of hand-dissected Drosophila mature oocytes (containing ~540 µg of total RNA) were subjected to separation by velocity sedimentation through sucrose gradients. In this way, free mRNAs (present in RNPs fraction) or those comigrating with ribosomal subunits (40S or 60S+80S fractions) or with varying numbers of bound ribosomes (low polysomes (2-4 ribosomes), medium polysomes (5-9 ribosomes), and heavy polysomes (more than 10 ribosomes) can be separated based on their size and collected as sucrose gradient fractions. To compare quantitatively the levels of every mRNA across the polysome gradient fractions, we added 5ng of S. cerevisiae mRNA as an exogenous spike-in to each of the six fractions of interest: RNPs, 40S, 60S+80S, low polysomes, medium polysomes and heavy polysomes. RNA was extraced from these fractions, follwing proteinase K treatment, by hot acid phenol method. In case of unfractionated lysates, RNA was extracted using TRIzol (Invitrogen) according to manufacturer’s instructions. mRNA-seq samples were prepared from 1 µg of total RNA (in case of sucrose gradient fractions and unfractionated lysates) and subject to Illumina based sequencing. Puromycin-treated lysates of mature oocytes or 0-2h Drosophila activated eggs (containing ~540 µg of total RNA) were also subjected to separation by velocity sedimentation through sucrose gradients. Puromycin causes premature termination of elongating ribosomes and thus it can be used to determine whether the mRNAs co-sedimenting with the polysomal peaks (defined here as =5 ribosomes) were actively engaged in translation. As an independent approach to assess translation and obtain information on the position of ribosomes on mRNAs, we employed ribosome footprinting. In addition to analyzing the same samples, as by polysome profiling, we also analyzed png mutant activated eggs by ribosome footprinting. Ribosome footprint profiling measures the number of ribosome-protected fragments (RPFs) derived from the mRNAs of each gene, resulting in a singular value of translational efficiency (TE) for each gene (TE=RPF/RNA).
Publication Title
Widespread changes in the posttranscriptional landscape at the Drosophila oocyte-to-embryo transition.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Danio rerio
- 7 Downloadable Samples
- IlluminaGenomeAnalyzerII
Description
RNA libraries from immunoprecipitates of Tdrd1, Ziwi and Zili, total testis RNA, total RNA from 3 week old wild-type and tdrd1 mutant gonads. Overall design: Both size selected and non-size selected libraries were made. Sequencing was performed using Illumina platform.
Publication Title
Tdrd1 acts as a molecular scaffold for Piwi proteins and piRNA targets in zebrafish.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 20 Downloadable Samples
- Illumina HiSeq 2000
Description
This dataset is part of a study that investigated how the hematopoietic system coordinates the rapid and efficient regeneration of the megakaryocytic lineage during stress scenarios. We found that the phenotypic hematopoietic stem cell (HSC) compartment contains stem-like megakaryocyte-committed progenitors (SL-MkPs), a cell population that shares many features with multipotent HSCs and serves as a lineage-restricted emergency pool for inflammatory insults. This dataset contains single-cell RNA sequencing data of 30 hematopoietic stem and progenitor cells which, in the context of our study, confirmed that MK-specfic transcripts are of highly variable expression in HSCs. The dataset further showed that variations in MK transcript expression in HSCs is not correlated with global transcriptomic rearrangements. Overall design: Murine bone marrow cells were sorted by Lin-cKit+CD150+CD48- (referred to as cd150+ in the following) and Lin-cKit+CD150- (referred to as cd150- in the following). Transcriptomes of 11 cd150- and 9 cd150+ HSCs were determined using QUARTZ, a single-cell RNASeq protocol
Publication Title
Inflammation-Induced Emergency Megakaryopoiesis Driven by Hematopoietic Stem Cell-like Megakaryocyte Progenitors.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 6 Downloadable Samples
Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Reduced chromatin binding of MYC is a key effect of HDAC inhibition in MYC amplified medulloblastoma.
Sample Metadata Fields
Specimen part, Treatment
View Samples- Homo sapiens
- 6 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
MYC is a driver oncogene in many cancers. Inhibition of MYC promises high therapeutic potential, but specific MYC inhibitors remain unavailable for clinical use. Previous studies suggest that MYC amplified Medulloblastoma cells are vulnerable to HDAC inhibition. Using co-immunoprecipitation, mass spectrometry and ChIP-sequencing we show that HDAC2 is a cofactor of MYC in MYC amplified primary medulloblastoma and cell lines. The MYC-HDAC2 complex is bound to genes defining the MYC-dependent transcriptional profile. Class I HDAC inhibition leads to stabilization and reduced DNA binding of MYC protein inducing a down-regulation of MYC activated genes (MAGs) and up-regulation of MYC repressed genes (MRGs). MAGs and MRGs are characterized by opposing biological functions and distinct E-box distribution. We conclude that MYC and HDAC2 (class I) are localized in a complex in MYC amplified medulloblastoma and drive a MYC-specific transcriptional program, which is reversed by the class I HDAC inhibitor entinostat. Thus, the development of HDAC inhibitors for treatment of MYC amplified medulloblastoma should include HDAC2 in its profile in order to directly target MYC´s trans-activating and trans-repressing function.
Publication Title
Reduced chromatin binding of MYC is a key effect of HDAC inhibition in MYC amplified medulloblastoma.
Sample Metadata Fields
Specimen part, Treatment
View Samples- Homo sapiens
- 15 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
High anaplastic lymphoma kinase (ALK) protein levels may be correlated with an unfavorable prognosis in neuroblastoma (NBL) patients, regardless of ALK mutation status. We therefore examined the correlation between levels of ALK, phosphorylated ALK (pALK) and downstream signaling proteins and response to ALK inhibition in a large panel of both ALK mutated (MUT) and wild type (WT) NBL cell lines. Six of the nineteen NBL cell lines had a point mutation and four an amplification of the ALK gene. ALK amplified cell lines showed similar ALK levels and ALK inhibitor sensitivity as WT cell lines and were therefore co-analyzed. The ALK mRNA (p=0.043), ALK 220 kDa (p=0.009) and ALK 140 kDa (p=0.025) protein levels were higher in ALK mutant (n=6) than WT cell lines (n=13). ALK mRNA and protein levels significantly correlated with ERK1 and ERK2 protein levels, and also with PHOX2B mRNA levels, a neural differentiation marker which is mutated in NBL. Response to ALK inhibitor TAE684 was also significantly correlated with ALK levels. ALK mutant cell lines (n=4) demonstrated a higher sensitivity towards ALK inhibitor TAE684 (14.9 fold more sensitive, p=0.004) than eight WT cell lines. These results underline the importance of ALK mutations but also ALK levels for response to ALK inhibitors in NBL cell lines. Furthermore, the strong correlation of PHOX2B and ALK suggests that neural differentiation stage may be correlated with ALK levels in neuroblastoma. These data will enhance understanding of ALK inhibitor response in future patient trials.
Publication Title
Anaplastic lymphoma kinase (ALK) inhibitor response in neuroblastoma is highly correlated with ALK mutation status, ALK mRNA and protein levels.
Sample Metadata Fields
Sex, Specimen part
View Samples- Homo sapiens
- 14 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
BCAT1 restricts αKG levels in AML stem cells leading to IDHmut-like DNA hypermethylation.
Sample Metadata Fields
Cell line, Treatment
View Samples- Homo sapiens
- 14 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
The branched chain amino acid (BCAA) pathway and high levels of BCAA transaminase 1 (BCAT1) have recently been associated with aggressiveness in several cancer entities. However, the mechanistic role of BCAT1 in this process remains largely uncertain. By performing high-resolution proteomic analysis of human acute myeloid leukaemia (AML) stem cell (LSC) and non-LSC populations, we found the BCAA pathway enriched and BCAT1 overexpressed in LSCs. We show that BCAT1, which transfers -amino groups from BCAAs to -ketoglutarate (KG), is a critical regulator of intracellular KG homeostasis. Next to its role in the tricarboxylic acid (TCA) cycle KG is an essential co-factor for KG-dependent dioxygenases such as EGLN1 and the TET family of DNA demethylases. Knockdown of BCAT1 in leukaemia cells caused accumulation of KG leading to HIF1a protein degradation mediated by EGLN1. This resulted in a growth and survival defect and abrogated leukaemia-initiating potential. In contrast, overexpression (OE) of BCAT1 in leukaemia cells decreased intracellular KG levels and caused DNA hypermethylation via altered TET activity. BCAT1high AMLs displayed a DNA hypermethylation phenotype similar to cases carrying mutant isocitrate dehydrogenase (IDHmut), in which TET2 is inhibited by the oncometabolite 2-hydroxyglutarate. High levels of BCAT1 strongly correlate with shorter overall survival in IDHwtTET2wt, but not IDHmut or TET2mut AMLs. Gene sets characteristic for IDHmut AMLs were enriched in IDHwtTETwtBCAT1high patient samples. BCAT1high AMLs showed robust enrichment for LSC signatures and paired sample analysis revealed a significant increase of BCAT1 levels upon disease relapse. In summary, by limiting intracellular KG, BCAT1 links BCAA catabolism to HIF1a stability and regulation of the epigenomic landscape. Our results suggest the BCAA-BCAT1-KG pathway as a therapeutic target to compromise LSC function in IDHwtTET2wt AML patients.
Publication Title
BCAT1 restricts αKG levels in AML stem cells leading to IDHmut-like DNA hypermethylation.
Sample Metadata Fields
Treatment
View Samples- Homo sapiens
- 14 Downloadable Samples
- Affymetrix Human Genome U133A Array (hgu133a)
Description
B cells from human tonsil and blood were sorted using flow cytometry. The human samples were processed immediately ex-vivo using markers for known B cell subsets.
Publication Title
Analysis of somatic hypermutation in X-linked hyper-IgM syndrome shows specific deficiencies in mutational targeting.
Sample Metadata Fields
No sample metadata fields
View Samples
GSE12366- Homo sapiens
- 10 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Sorted B cells using flow cytometry
Publication Title
Analysis of somatic hypermutation in X-linked hyper-IgM syndrome shows specific deficiencies in mutational targeting.
Sample Metadata Fields
No sample metadata fields
View Samples