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SRP148097
Homo sapiens
12 Downloadable Samples
Illumina HiSeq 2500
Description
Quiescent stem cells of glioblastoma (GBM), a malignant primary brain tumor, are potential sources for recurrence after therapy. However, the gene expression program underlying the physiology of GBM stem cells remains unclear. We have isolated quiescent GBM cells by engineering them with a knock-in H2B-GFP proliferation reporter and expanding them in a 3D tumor organoid model that mimics tumor heterogeneity. H2B-GFP label retaining quiescent cells were subjected to stem cell assays and RNA-Seq gene expression analysis. While quiescent GBM cells were similar in clonal culture assays to their proliferative counterparts, they displayed higher therapy resistance. Interestingly, quiescent GBM cells upregulated epithelial-mesenchymal transition (EMT) genes and genes of extracellular matrix components. Our findings connect quiescent GBM cells with an EMT-like shift, possibly explaining how GBM stem cells achieve high therapy resistance and invasiveness, and suggest new targets to abrogate GBM. Overall design: Glioblastoma cancer cells in 3D organoid culture were pulsed for 2 weeks with H2B-GFP, then chased either 2 or 4 weeks. Label-retaining GFP-high cells (quiescent) were separated from bulk population, and both populations were analyzed by RNA-Seq.
Publication Title
Gene signatures of quiescent glioblastoma cells reveal mesenchymal shift and interactions with niche microenvironment.
Sample Metadata Fields
Specimen part, Subject
View Samples- Homo sapiens
- 2 Downloadable Samples
- Illumina HiSeq 2500
Description
Studying dynamic transcripts in single cells (SC) requires large numbers of timed samples. We report an easy to use protocol to stabilize RNA in intact SCs without perturbing transcriptional patterns, and demonstrate its applicability for SC transcriptome assays with cells and tissue. We identify a gene-specific hierarchical pattern of all-or-none transcript induction elicited by different concentrations of pulsatile hormone stimuli in pituitary gonadotropes. Overall design: SC Mini Drop-seq experiments were performed on two samples from dissociated human cortical tissue: one is neocortex from a glioblastoma multiforme (GBM, tumor), the other is normal neocortex adjacent to the tumor.
Publication Title
Single-cell stabilization method identifies gonadotrope transcriptional dynamics and pituitary cell type heterogeneity.
Sample Metadata Fields
Specimen part, Subject
View Samples- Homo sapiens
- 21 Downloadable Samples
Affymetrix Human Genome U133A Array (hgu133a)
Description
The purpose of the experiment was to generate a time course of gene expression following irradiation. The goal was then to model this data to extract hidden variables - chiefly, the activity profiles of the p53 transcription factor. Using this information the aim was to predict which transcripts changed by IR were targets of p53. Cells in log phase (1 x 106 ml-1) were ?-irradiated with 5 Gy at room temperature (RT) at a dose rate of 2.45 Gy per minute with a 137Cs ?-irradiator. Cells were harvested at 0, 2, 4, 6, 8, 10 and 12 hours, and RNA and protein were extracted (Trizol, Invitrogen). Affymetrix U133A arrays were hybridized as standard (www.affymetrix.co.uk). Array quality was determined using R and GCOS .rpt file values. The time course was replicated three times from independent cell preparations.
Publication Title
Ranked prediction of p53 targets using hidden variable dynamic modeling.
Sample Metadata Fields
Specimen part, Disease, Cell line, Time
View Samples- Rattus norvegicus
- 7 Downloadable Samples
- Affymetrix Rat Genome 230 2.0 Array (rat2302), UNKNOWN
Description
A study of diabetic neuropathy in dorsal root ganglia from streptozotocin-diabetic male wistar rats over the first 8 weeks of diabetes
Publication Title
Identification of changes in gene expression in dorsal root ganglia in diabetic neuropathy: correlation with functional deficits.
Sample Metadata Fields
Sex, Age, Specimen part, Disease, Disease stage, Time
View Samples- Mus musculus
- 7 Downloadable Samples
- NextSeq 500
Description
Identification of temporal changes in gene expression in macrophages isolated from the site of nerve injury. Overall design: Macrophages were profiled at 3 timepoints (5, 14, and 28 days) after nerve injury with 2-3 independent biological replicates per timepoint.
Publication Title
Temporal changes in macrophage phenotype after peripheral nerve injury.
Sample Metadata Fields
Subject, Time
View Samples- Homo sapiens
- 6 Downloadable Samples
- Illumina HiSeq 2000
Description
Type 1 diabetes (T1D) is caused by autoimmune destruction of pancreatic ß cells. Mounting evidence supports a central role for ß-cell alterations in triggering the activation of self-reactive T-cells in T1D. However, the early deleterious events that occur in ß cells, underpinning islet autoimmunity are not known. We hypothesized that epigenetic modifications induced in ß cells by inflammatory mediators play a key role in initiating the autoimmune response. We analyzed DNA methylation (DNAm) patterns and gene expression in human islets exposed to IFNa, a cytokine associated with T1D development. We found that IFNa triggers DNA demethylation and increases expression of genes controlling inflammatory and immune pathways. We then demonstrated that DNA demethylation was caused by up-regulation of the exoribonuclease, PNPase Old-35 (PNPT1), which caused degradation of miR-26a. This in turn promoted the up-regulation of ten-eleven translocation TET2 enzyme and increased 5-hydoxymethylcytosine levels in human islets and pancreatic ß-cells. Moreover, we showed that specific IFNa expression in the ß cells of IFNa-INS1CreERT2 transgenic mice, led to development of T1D that was preceded by increased islet DNA hydroxymethylation through a PNPT1/TET2-dependent mechanism. Our results suggest a new mechanism through which IFNa regulates DNAm in ß cells, leading to changes in expression of genes in inflammatory and immune pathways that can initiate islet autoimmunity in T1D. Overall design: We exposed human pancreatic islets from three donors to 2000 IU IFNa and assessed gene expression by RNAseq. The cDNA library was prepared using Illumina TruSeq RNA Sample Prep Kits. Next generation sequencing was performed on Illumina HiSeq2000 using the Single-Read Cluster Generation kit v2 and SBS Sequencing kit v3. Image analysis and base calling were conducted using the SDS 2.5/RTA1.5 software.
Publication Title
Epigenetic modulation of β cells by interferon-α via PNPT1/mir-26a/TET2 triggers autoimmune diabetes.
Sample Metadata Fields
Specimen part, Disease stage, Treatment, Subject
View Samples- Mus musculus
- 5 Downloadable Samples
- Illumina MouseWG-6 v2.0 expression beadchip
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation.
Sample Metadata Fields
Specimen part, Cell line
View Samples- Homo sapiens
- 11 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Cell-and context-specific activities of nuclear receptors may in part be due to distinct coregulator complexes recruited to distinct subsets of target genes. RIP140 (also called NRIP1) is a ligand-dependent corepressor that is inducible with retinoic acid (RA). We have shown previously that silencing of RIP140 enhances RA-induced differentiation and enhances the induction of model RA target genes in human embryonal carcinoma cells (EC). Through use of microarray technology we sought to elucidate in a de novo fashion the global role of RIP140 in RA-dependent signaling. RIP140-dependent gene expression was largely consistent with RIP140 functioning to limit RAR signaling. Few if any genes were regulated in a manner to support a role for RIP140 in active repression. Interestingly, approximately half of the RA-dependent genes were unaffected by RIP140, suggesting that RIP140 may discriminate between different classes of RA target genes. RIP140 silencing also accelerated RA target gene activation and sensitized EC cells to low doses of RA. Together the data suggests that the RIP140-dependent RA target genes identified here may be particularly important in mediating RA-induced tumor cell differentiation. RIP140 may be an attractive target to sensitize tumor cells to retinoid-based differentiation therapy.
Publication Title
Selective repression of retinoic acid target genes by RIP140 during induced tumor cell differentiation of pluripotent human embryonal carcinoma cells.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 5 Downloadable Samples
- Illumina MouseWG-6 v2.0 expression beadchip
Description
Self-renewal of embryonic stem cells (ESCs) cultured in serum-LIF is incomplete with some cells initiating differentiation. While this is reflected in heterogeneous expression of naive pluripotency transcription factors (TFs), the link between TF heterogeneity and differentiation is not fully understood. Here we purify ESCs with distinct TF expression levels from serum-LIF cultures to uncover early events during commitment from nave pluripotency. ESCs carrying fluorescent Nanog and Esrrb reporters show Esrrb downregulation only in NANOGlow cells. Independent Esrrb reporter lines demonstrate that ESRRBnegative ESCs cannot effectively self-renew. Upon ESRRB loss, pre-implantation pluripotency gene expression collapses. ChIP-Seq identifies different regulatory element classes that bind both OCT4 and NANOG in ESRRBhigh cells. Class I elements lose NANOG and OCT4 binding in ESRRBnegative ESCs and associate with genes expressed preferentially in nave ESCs. In contrast, class II elements retain OCT4 but not NANOG binding in ESRRBnegative cells and associate with more broadly expressed genes. Therefore, mechanistic differences in TF function act cumulatively to restrict potency during exit from nave pluripotency.
Publication Title
Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation.
Sample Metadata Fields
Specimen part
View Samples- Homo sapiens
- 6 Downloadable Samples
Description
The exosome-independent exoribonuclease DIS3L2 is mutated in Perlman syndrome. Here we used extensive global transcriptomic and targeted biochemical analyses to identify novel DIS3L2 substrates in human cells. We show that DIS3L2 regulates pol II transcripts, comprising selected canonical and histone-coding mRNAs, and a novel FTL_short RNA from the ferritin mRNA 5'' UTR. Importantly, DIS3L2 contributes to surveillance of pre-snRNAs during their cytoplasmic maturation. Among pol III transcripts, DIS3L2 particularly targets vault and Y RNAs and an Alu-like element BC200 RNA, but not Alu repeats, which are removed by exosome-associated DIS3. Using 3'' RACE-Seq, we demonstrate that all novel DIS3L2 substrates are uridylated in vivo by TUT4/TUT7 poly(U) polymerases. Uridylation-dependent DIS3L2-mediated decay can be recapitulated in vitro, thus reinforcing the tight cooperation between DIS3L2 and TUTases. Together these results indicate that catalytically inactive DIS3L2, characteristic of Perlman syndrome, can lead to deregulation of its target RNAs to disturb transcriptome homeostasis. Overall design: To investigate DIS3L2 functions genome-wide, total RNA samples were collected from model cell lines producing either WT or mut DIS3L2 three days after induction with doxycycline. The RNA samples were rRNA-depleted before preparation of strand-specific total RNA libraries according to the standard TruSeq (Illumina) protocol. TruSeq library preparation favours RNA molecules longer than 200 nt, and shorter transcripts are suboptimal for sequencing via this protocol. Thus, to obtain information about potential DIS3L2 RNA substrates with lengths between 20 and 220 nt, another RNA-Seq was carried out in parallel (with size selection through gel purification). The stable inducible HEK293 cell lines producing DIS3L2 variants were obtained using “pAL_01” and “pAL_02” plasmid constructs and the Flp-In™ T-REx™ system according to the manufacturer’s guidelines. “pAL_01” and “pAL_02” plasmids are vectors for co-expression of recoded C-terminal FLAG-tagged DIS3L2 [wild type (WT) variant or its catalytic mutant counterpart (mut), respectively] and sh-miRNAs directed against endogenous DIS3L2 mRNA.
Publication Title
Perlman syndrome nuclease DIS3L2 controls cytoplasmic non-coding RNAs and provides surveillance pathway for maturing snRNAs.
Sample Metadata Fields
No sample metadata fields
View Samples