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Mus musculus
6 Downloadable Samples
Illumina Genome Analyzer IIx
Description
The earliest stages of Huntington’s disease are marked by changes in gene expression that are caused in an indirect and poorly understood manner by polyglutamine expansions in the huntingtin protein (HTT). To explore the hypothesis DNA methylation may be altered in cells expressing mutated HTT, we use reduced-representation bisulfite sequencing (RRBS) to map sites of DNA methylation in cells carrying either wild-type or mutant HTT. We find that a large fraction of the genes that change in expression in the presence of mutant huntingtin demonstrate significant changes in DNA methylation. Regions with low CpG content, which have previously been shown to undergo methylation changes in response to neuronal activity, are disproportionately affected. Based on the sequence of regions that change in methylation, we identify AP-1 and SOX2 as transcriptional regulators associated with DNA methylation changes, and we confirm these hypotheses using genome-wide chromatin immunoprecipitation (ChIP-Seq). Our findings suggest new mechanisms for the effects of polyglutamine-expanded HTT. These results also raise important questions about the potential effects of changes in DNA methylation on neurogenesis and at later stages, cognitive decline in Huntington’s patients. Overall design: mRNA-seq in STHdhQ7/Q7 and STHdhQ111/Q111 cells
Publication Title
Extensive changes in DNA methylation are associated with expression of mutant huntingtin.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples
SRP046257- Mus musculus
- 12 Downloadable Samples
- Illumina HiSeq 2000
Description
RNA-Seq performed on Dicer KO and WT murine mesenchymal stem cells from total RNA MicroRNAs (miRNAs) are small non-coding RNAs that regulates development and disease but induce only moderate repression of directs mRNA targets, suggesting that they coordinate with other modes ofs cellular regulation to effect large changes in gene expression. Ins this work we decouple direct effects of global miRNA loss froms transcriptional changes downstream in a pair of isogenic murines fibroblast cell lines with and without Dicer expression. Wes demonstrate how effects on direct miRNA targets are amplified bys transcription machinery through the construction of a network models that identifies specific transcription factors that cause changes ins mRNA expression upon Dicer loss. Through transcription factors over-expression, we delineate miRNA-mediated transcriptional programss and identify miRNA-mediated coherent and incoherent feed-forwards loops, suggesting a functional role of the interaction between miRNAss and transcription factors. In total, our results indicate thats miRNAs tightly control transcription factors within a denses interconnected network to modulate gene expression. Overall design: Total RNA was analyzed from adult mesenchymal stem cells (immortalized monoclonal lines of murine MSCs) with and without Dicer (WT: Dicer f/f, KO: Dicer -/-), as well as from WT cells transfected with an empty vector or a vector containing Tead4, Sox9 or Pbx3 transcripts.
Publication Title
Elucidating MicroRNA Regulatory Networks Using Transcriptional, Post-transcriptional, and Histone Modification Measurements.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 137 Downloadable Samples
- Illumina HiSeq 2500
Description
Purpose: To identify regulatory proteins that are potential drivers of a coordinated breast cancer metastasis gene expression signatures. Methods: Knockdown of target genes in breast cancer cell lines was achieved using scramble and/or gene-specific siRNA (ON-TARGET SMARTpool, Thermo Scientific) and Lipofectamine RNAiMAX. 48h post transfection, total RNA was isolated from cell lines using the RNeasy Plus mini prep kit (Qiagen). Nucleic acid quality was determined with the Agilent 2100 Bioanalyzer. RNA Sequencing was also performed at the New York Genome Center (Manhattan, NY, USA) using a HiSeq 2500 Ultra-High-Throughput Sequencing System (Illumina, San Diego, CA, USA). Results: Raw reads in the fastq format were aligned to Human Genome HG19 using the RNA-seq STAR aligner version 2.4.0d (http://www.ncbi.nlm.nih.gov/pubmed/23104886, http://www.ncbi.nlm.nih.gov/pubmed/26334920) as recommended by user manual downloaded along with the software. STAR aligner was chosen for mapping accuracy and speed (http://www.nature.com/nmeth/journal/v10/n12/full/nmeth.2722.html). Mapped reads for each sample were counted for each gene in annotation files in GTF format (gencode.v19.annotation.gtf available for download from GENECODE website (http://www.gencodegenes.org/releases/19.html)) using the FeatureCounts read summarization program (http://www.ncbi.nlm.nih.gov/pubmed/?term=24227677) following the user guide (http://bioinf.wehi.edu.au/subread-package/SubreadUsersGuide.pdf). Individual count files were merged to generate the raw-counts matrix by an in-house R script, normalized to account for differences in library size and the variance was stabilized by fitting the dispersion to a negative-binomial distribution as implemented in the DESeq R package (http://bioconductor.org/packages/release/bioc/html/DESeq.html)(Anders and Huber, 2010). Conclusions: Our data suggest that targeting keystone proteins in the breast cancer metastasis transcriptome can effectively collapse transcriptional hierarchies necessary for metastasis formation, thus representing a formidable cancer intervention strategy. Overall design: Examination of mRNA profiling of breast cancer cell lines after knock-down of putative master regulators of the breast cancer metastasis transcriptome
Publication Title
An Integrated Systems Biology Approach Identifies TRIM25 as a Key Determinant of Breast Cancer Metastasis.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Danio rerio
- 6 Downloadable Samples
Affymetrix Zebrafish Genome Array (zebrafish)
Description
Cone photoreceptors are specialised sensory retinal neurons responsible for photopic vision, colour perception and visual acuity. Retinal degenerative diseases are a heterogeneous group of eye diseases in which the most severe vision loss typically arises from cone photoreceptor dysfunction or degeneration. Establishing a method to purify cone photoreceptors from retinal tissue can accelerate the identification of key molecular determinants that underlie cone photoreceptor development, survival and function. The work herein describes a new method to purify enhanced green fluorescent protein (EGFP)-labelled cone photoreceptors from adult retina of Tg(3.2TCP:EGFP) zebrafish. Electropherograms confirmed downstream isolation of high-quality RNA with RNA integrity number (RIN) >7.6 and RNA concentration >5.7 ng/l obtained from both populations. Reverse Transcriptase-PCR (RT-PCR) confirmed that the EGFP-positive cell populations express known genetic markers of cone photoreceptors that were not expressed in the EGFP-negative cell population. This work is an important step towards the identification of cone photoreceptor-enriched genes, protein and signalling networks responsible for their development, survival and function. In addition, this advancement facilitates the identification of novel candidate genes for inherited human blindness.
Publication Title
HDAC6 inhibition by tubastatin A is protective against oxidative stress in a photoreceptor cell line and restores visual function in a zebrafish model of inherited blindness.
Sample Metadata Fields
Specimen part
View Samples- Rattus norvegicus
- 9 Downloadable Samples
- IlluminaGenomeAnalyzer
Description
MicroRNAs (miRNAs) are single-stranded non-coding RNAs that negatively regulate target gene expression through mRNA cleavage or translational repression. There is mounting evidence that they play critical roles in heart disease. The expression of known miRNAs in the heart has been studied at length by microarray and quantitative PCR but it is becoming evident that microRNA isoforms (isomiRs) are potentially physiologically important. It is well known that left ventricular (patho)physiology is influenced by transmural heterogeneity of cardiomyocyte phenotype, and this likely reflects underlying heterogeneity of gene expression. Given the significant role of miRNAs in regulating gene expression, knowledge of how the miRNA profile varies across the ventricular wall will be crucial to better understand the mechanisms governing transmural physiological heterogeneity. To determinine miRNA/isomiR expression profiles in the rat heart we investigated tissue from different locations across the left ventricular wall using deep sequencing. We detected significant quantities of 145 known rat miRNAs and 68 potential novel orthologs of known miRNAs, in mature, mature* and isomiR formation. Many isomiRs were detected at a higher frequency than their canonical sequence in miRBase and have different predicted targets. The most common miR-133a isomiR was more effective at targeting a construct containing a sequence from the gelsolin gene than was canonical miR-133a, as determined by dual-fluorescence assay. We identified a novel rat miR-1 homolog from a second miR-1 gene; and a novel rat miRNA similar to miR-676. We also cloned and sequenced the rat miR-486 gene which is not in miRBase (v18). Signalling pathways predicted to be targeted by the most highly detected miRNAs include Ubiquitin-mediated Proteolysis, Mitogen-Activated Protein Kinase, Regulation of Actin Cytoskeleton, Wnt signalling, Calcium Signalling, Gap junctions and Arrhythmogenic Right Ventricular Cardiomyopathy. Most miRNAs are not expressed in a gradient across the ventricular wall, with exceptions including miR-10b, miR-21, miR-99b and miR-486. Overall design: The hearts of 3 male 8 month old Sprague-Dawley rats were rapidly extracted after euthanasia with sodium pentobarbital. A section of the free wall of the left ventricle was dissected into epicardium, mid-myocardium and endocardium by cutting approximately 1 mm from the epicardial and endocardial surfaces. Small RNA was extracted (miRNeasy Kit; Qiagen, Crawley UK), quantified (Nanodrop; Thermo Scientific) and quality assessed for degradation (RNA Nano Chip, Bioanalyser 2100; Aligent Technologies, Wokingham UK; only samples with a RNA integrity no. (RIN) =8 were carried forward) and retention of small RNA (Small RNA Chip, Bioanalyser 2100). Small RNA was preferentially ligated with adapters, reverse transcribed into cDNA and amplified with 9 individually tagged primer indices (TruSeq Small RNA Sample Preparation Kit; Illumina, Little Chesterford, UK) and a library of small RNA created for each sample. After gel purification the cDNA products were again analysed on the bioanalyser using a High Sensitivity DNA Chip and assessed for the presence and concentration of the peak corresponding to ligated and tagged miRNA (approximately 147nt). Only samples with suitable RIN values exhibiting good retention of small RNA species were used for library preparation. After pooling, the samples were sequenced by TrinSeq (Trinity Genome Sequencing Lab & Neuropsychiatric Genetics Group, Trinity College Dublin, Ireland (http://www.medicine.tcd.ie/sequencing); using TruSeq SR Cluster Kit v5 (Illumina) and the resultant data trimmed and aligned to miRBase v18 (CLC Genomics Workbench v4.0; CLC bio, Swansea UK).
Publication Title
Distinctive profile of IsomiR expression and novel microRNAs in rat heart left ventricle.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 8 Downloadable Samples
- Illumina HiSeq 4000
Description
TRAP-seq gene expression profiling. GFP-L10a is expressed in BDNF+ or PACAP+ in preoptic area. Bdnf+ or Pacap+ cells in the preoptic area are labelled with GFP-L10a fusion protein expression to study cell-type specific gene expression. We used adult BDNF-Cre (custom generated, discussed in submitted paper) and Pacap-Cre mice (Allen Brain Institute, gift Hongkui Zeng) and injected Cre-depndent adenoassociated virus (AAV2-EF1a-DIO-EGFP-L10a, custom made) into the ventromedial preoptic area. Overall design: Comparison of Immunoprecipitation and Input fraction mRNA
Publication Title
Warm-Sensitive Neurons that Control Body Temperature.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Mus musculus
- 6 Downloadable Samples
- Illumina HiSeq 2500
Description
RNA-Seq profiling identifies transcripts enriched in preoptic neurons activated by warm temperature challenge Overall design: Animals are subject to warm temperatures during which ribosomal S6 protein is phosphorylated selectively in active neurons. Transcripts associated with phosphorylated ribosome is isolated by immunoprecipitation. Comparison of Immunoprecipitation and Input fraction mRNA
Publication Title
Warm-Sensitive Neurons that Control Body Temperature.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Homo sapiens
- 32 Downloadable Samples
- Illumina HiSeq 2500
Description
Differentiation events contribute to cellular heterogeneity within tumors and influence disease progression and response to therapy. Here we dissect the mechanisms controlling intratumoral heterogeneity within basal-like breast cancers. We show that cancer cells can transition between a differentiation state related to that of normal luminal progenitors and a state closer to that of mature luminal cells, and that this occurs through asymmetric cell divisions. The Polycomb factor EZH2 and the Notch pathway act to increase the rates of symmetric divisions that produce progenitor-like cells, while the FOXA1 transcription factor promotes asymmetric divisions that reduce the numbers of such cells. Through functional screening, we identified a group of regulators that control cancer cell differentiation state and the relative proportions of tumor cell subpopulations. Our findings highlight the regulation of asymmetric cell divisions as a mechanism controlling intratumoral heterogeneity, and identify molecular pathways that control breast cancer cellular composition. Overall design: Expression profiles of HCC70 cells expressing shRNAs targeting regulatory factors that influence basal-like cancer cell population composition
Publication Title
Regulation of Cellular Heterogeneity and Rates of Symmetric and Asymmetric Divisions in Triple-Negative Breast Cancer.
Sample Metadata Fields
Cell line, Subject
View Samples- Homo sapiens
- 6 Downloadable Samples
- Illumina HiSeq 2500
Description
Differentiation events contribute to cellular heterogeneity within tumors and influence disease progression and response to therapy. Here we dissect the mechanisms controlling intratumoral heterogeneity within basal-like breast cancers. We show that cancer cells can transition between a differentiation state related to that of normal luminal progenitors and a state closer to that of mature luminal cells, and that this occurs through asymmetric cell divisions. The Polycomb factor EZH2 and the Notch pathway act to increase the rates of symmetric divisions that produce progenitor-like cells, while the FOXA1 transcription factor promotes asymmetric divisions that reduce the numbers of such cells. Through functional screening, we identified a group of regulators that control cancer cell differentiation state and the relative proportions of tumor cell subpopulations. Our findings highlight the regulation of asymmetric cell divisions as a mechanism controlling intratumoral heterogeneity, and identify molecular pathways that control breast cancer cellular composition. Overall design: Expression profiles of three cell subpopulations – K18+, K18+K14+ and K18+Vim+ – sorted from the breast cancer cell lines HCC70 and MDA-MB-468
Publication Title
Regulation of Cellular Heterogeneity and Rates of Symmetric and Asymmetric Divisions in Triple-Negative Breast Cancer.
Sample Metadata Fields
Cell line, Subject
View Samples- Arabidopsis thaliana
- 18 Downloadable Samples
- Affymetrix Arabidopsis ATH1 Genome Array (ath1121501)
Description
Arabidopsis thaliana cell suspension cultures (ACSC) were subjected to 30-min, mild chemical treatments with three different singlet oxygen elicitors at low-medium light conditions (150 E m2 s1) with the aim of getting a better understanding of singlet oxygen-mediated defence responses in plants. The three elicitors Indigo Carmine (IC), Methylene Violet (MV) and Rose Bengal (RB) at a concentration of 0.5 M were chosen because they exhibited different abilities to permeate the plasma membrane and to accumulate in the cell soma or organelles such as chloroplasts. In addition, ACSC were treated with 500 M H2O2 for comparison. Confocal image analysis of Arabidopsis cells revealed that IC was not retained in cells, whereas MV and RB permeated the plasma membrane and accumulated in the chloroplast envelope and inside chloroplasts, respectively. As a consequence of their different cellular location, the physiological, transcriptional and photosynthetic responses of Arabidopsis cells to singlet oxygen production varied from each other and the activation of programmed cell death (PCD) was observed in ACSC treated with 0.5 M RB, but not with the other elicitor nor with 500 M H2O2. The role of chloroplasts in the activation of PCD was further investigated when this physiological response was analyzed in dark-grown cell cultures containing undifferentiated plastids. Interestingly, PCD was only activated in light-grown, but not in dark-grown, Arabidopsis cell cultures, suggesting that singlet oxygen-mediated defence responses were initiated inside chloroplasts. Genome-wide transcriptional profile analyses were performed as well and the results proved that there were only statistically significant changes in the transcript expression of light-grown ACSC treated with 0.5 M RB and 500 M H2O2, but not with IC nor with MV. Functional enrichment analyses revealed that GO/Biological process terms associated with defence responses were common in the treatments with 0.5 M RB and 500 M H2O2; however, resistance response to pathogen and PCD terms were only significantly over-represented in the RB treatment. Moreover, the analysis of the up-regulated transcripts in ACSC treated with 0.5 M RB brought out that both specific markers for singlet oxygen from the conditional fluorescence (flu) mutant of Arabidopsis and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present, although there was no evidence for the up-regulation of EDS1 encoding the ENHANCED DISEASE SUSCEPTIBILITY PROTEIN 1. Finally, a co-regulation analysis proved that ACSC treated with 0.5 M RB exhibited higher correlation with the flu family mutants than with other singlet oxygen producer mutants of Arabidopsis or wild-type plants of Arabidopsis subjected to high light treatments, where singlet oxygen was produced in photosystem II and an acclimatory response was activated instead of PCD.
Publication Title
Programmed cell death activated by Rose Bengal in Arabidopsis thaliana cell suspension cultures requires functional chloroplasts.
Sample Metadata Fields
Treatment
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