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Mus musculus
8 Downloadable Samples
Illumina HiSeq 2500
Description
The aim of this study is to evaluate the effect of Autoimmune regulator (Aire) gene disruption in a murine medullary thymic epithelial cells (mTEC 3.10 cell line) on the transcriptome of these cells during its adhesion with thymocytes. The mTEC-thymocyte adhesion is a crucial step for the negative selection of autoreactive thymocytes and prevention of autoimmune diseases. To generate Aire mutant cell clones, a total of 5x10^5 mTEC 3.10 cells were electro-transfected (Lonza Nucleofector) with CRISPR-Cas9 plasmid targeting the Aire Exon 3 (plasmid "all in one" encoding Aire Exon 3 gRNA + Cas9 + GFP, from Sigma-Aldrich). The GFP positive mTEC single cells were sorted by using a FACS Aria III cytometer and cells were cloned by expansion in culture. Sanger sequencing of PCR products from the Aire Exon 3 of these clones was used in order to evaluate the occurrence of indel mutations within the targeted Exon 3. The mTEC 3.10 clone E6 was identified and validated as a compound heterozygous Aire KO (Aire +/-). This clone features the Aire allele 1 that encodes a mutant Aire protein carring a neutral aminoacid substitution (A118P) and allele 2 encoding a truncated Aire protein. Wild type (WT) mTEC 3.10 cells or mTEC 3.10 clone E6 were cultured in the presence (or not) of thymocytes in order to establish cell adhesion. The total RNA preparations from WT or clone E6 mTEC cells (before or after mTEC- thymocyte co-cultures) were then sequenced through RNA-sequencing using a Illumina HiSeq 2500 instrument and the TruSeq Stranded mRNA Library Preparation kit resulting in about 50 million paired-end stranded specific 100 bp reads per sample. Sequencing reads were mapped to Mus musculus reference genome (mm10) using STAR v.2.5.0a. Read counts over transcripts were calculated using HTSeq v.0.6.1p2 based on a current UCSC annotation file for GRCm38/mm10 (Dec. 2011). Overall design: The mRNA profiles of mTEC 3.10 cells carring WT Aire (before or after co-culture with thymocytes) or heterozygous KO mTEC 3.10 cells (clone E6, Aire +/-) (before or after co-culture with thymocytes) were generated by sequencing, in duplicates, using a Illumina HiSeq 2500 instrument.
Publication Title
Aire Disruption Influences the Medullary Thymic Epithelial Cell Transcriptome and Interaction With Thymocytes.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Homo sapiens
- 35 Downloadable Samples
- IlluminaHiSeq2000
Description
The goal of this study is to simultaneously examine host and parasite gene expression programs in skin lesions of human patients infected with the intracellular parasite Leishmania. We conducted high-resolution sequencing of the transcriptomes from early and late stage cutaneous leishmaniasis biopsies using an RNA-seq approach. An array of computational tools was applied to map reads to the Leishmania and human genomes and reconstruct full-length transcripts. mRNA abundance was determined for Leishmania and human genes, helping to explain tuning of the immune response to parasite transcriptomic profiles present in the lesion microenvironment. This data provided a deeper look at the transcriptomic profile of the host response in conjunction with a novel look at the parasite transcriptome in human cutaneous lesions. These data also offer the first glimpse of Leishmania gene expression profiles specific to the cutaneous manifestation of disease in human patients. This metatranscriptomic study provides a solid framework for future functional, genomic, and clinical studies of leishmaniasis as well as intracellular pathogenesis in general.
Publication Title
Meta-transcriptome Profiling of the Human-Leishmania braziliensis Cutaneous Lesion.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 13 Downloadable Samples
Affymetrix Human Gene 1.0 ST Array (hugene10st)
Description
Non-syndromic cleft lip/palate (NSCL/P) is a complex, frequent congenital malformation, determined by the interplay between genetic and environmental factors during embryonic development. Previous findings have appointed an aetiological overlap between NSCL/P and cancer, and alterations in similar biological pathways may underpin both conditions. Here, using a combination of transcriptomic profiling and functional approaches, we report that NSCL/P dental pulp stem cells exhibit dysregulation of a co-expressed gene network mainly associated with DNA double-strand break repair and cell cycle control (p = 2.88x10-2 5.02x10-9). This network included important genes for these cellular processes, such as BRCA1, RAD51, and MSH2, which are predicted to be regulated by transcription factor E2F1. Functional assays support these findings, revealing that NSCL/P cells accumulate DNA double-strand breaks upon exposure to H2O2. Furthermore, we show that E2f1, Brca1 and Rad51 involved in DNA repair are co-expressed in the developing embryonic orofacial primordia, and may act as a molecular hub playing a role in lip and palate morphogenesis. In conclusion, we show that cellular defences against DNA damage may take part in the pathogenesis of NSCL/P, in accordance with the hypothesis of aetiological overlap between this malformation and cancer. These results provide more information regarding the aetiology of NSCL/P and have the potential tocan potentially assist incontribute to the development of future preventive strategies.
Publication Title
Susceptibility to DNA damage as a molecular mechanism for non-syndromic cleft lip and palate.
Sample Metadata Fields
Sex, Specimen part
View Samples- Mus musculus
- 12 Downloadable Samples
- Affymetrix Mouse Gene 1.0 ST Array (mogene10st)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
An RB-EZH2 Complex Mediates Silencing of Repetitive DNA Sequences.
Sample Metadata Fields
Specimen part
View Samples- Mus musculus
- 12 Downloadable Samples
- Affymetrix Mouse Gene 1.0 ST Array (mogene10st)
Description
Upon G1-S transition, cyclin-dependent kinases (CDKs) phosphorylate the retinoblastoma tumor suppressor protein (pRB) to release E2F transcription factors, which activate transcriptional programs, required for S-phase entry. Beyond the G1-S transition, pRB activity remains poorly understood. Our lab has discovered that pRB retains exclusive binding to E2F1 through an alternate E2F1-specific binding site at the pRB c-terminus independent of CDK phosphorylation. We have developed a gene-targeted mouse model that is defective for the E2F1-specific interaction. We are exploring the function of this complex through genome-wide binding and expression profiling. Overall, this work suggests an alternate pRB-E2F1 complex persists independent of CDK phosphorylation to establish regions of constitutive heterochromatin
Publication Title
An RB-EZH2 Complex Mediates Silencing of Repetitive DNA Sequences.
Sample Metadata Fields
Specimen part
View Samples
SRP048623- Mus musculus
- 12 Downloadable Samples
- Illumina HiSeq 2500
Description
Upon G1-S transition, cyclin-dependent kinases (CDKs) phosphorylate the retinoblastoma tumor suppressor protein (pRB) to release E2F transcription factors, which activate transcriptional programs, required for S-phase entry. Beyond the G1-S transition, pRB activity remains poorly understood. Our lab has discovered that hyperphosphorylated pRB (ppRB), found beyond G1, retains exclusive binding to E2F1 through an alternate E2F1-‘specific’ binding site at the pRB c-terminus. We have developed a gene-targeted mouse model that is defective for the E2F1-‘specific’ interaction. We are exploring the function of this complex through genome-wide expression profiling. Overall, this work suggests an alternate pRB-E2F1 complex persists beyond the G1-S transition to establish regions of constitutive heterochromatin. Overall design: 1. Total RNA from passage 4 quiescent MEFs isolated using TRIzol RNA extraction protocol 2. rRNA was depleted from total RNA using the RiboMinus Euk System V2 protocol according to manufacturer’s procotol 3. rRNA-depleted RNA samples were submitted for picoanalyzer analysis to determine concentration, purity, and rRNA content 4. Three wild-type and three mutant RNA samples with <10% rRNA remaining were submitted for library construction 5. Library was used for Illumina HiSeq 2500 paired end sequencing.
Publication Title
An RB-EZH2 Complex Mediates Silencing of Repetitive DNA Sequences.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 9 Downloadable Samples
- Affymetrix Mouse Gene 1.0 ST Array (mogene10st)
Description
The retinoblastoma protein (pRB) is best known for regulating cell proliferation through E2F transcription factors. In this report we investigate the properties of a targeted mutation that disrupts pRB interactions with the transactivation domain of E2Fs. Mice that carry this mutation endogenously (Rb1G) are defective in regulating E2F target genes. Surprisingly, cell cycle regulation in Rb1G/G MEFs strongly resembles that of wild type. In a serum deprivation induced cell cycle exit, Rb1G/G MEFs display a similar magnitude of E2F target gene derepression as Rb1-/-, even though Rb1G/G cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1G/G MEFs is responsive to p16 expression, indicating that the G-pRB protein can be activated in G1 to arrest proliferation through non-E2F mechanisms. Some Rb1G/G mice die neonatally with a muscle degeneration phenotype, while the others live a normal lifespan with no evidence of spontaneous tumor formation. Histological analysis reveals discrete examples of hyperplasia in the mammary epithelium, but most tissues appear normal while being accompanied by derepression of pRB regulated E2F targets. This suggests that non-E2F, pRB dependent pathways may have a more relevant role in proliferative control than previously identified.
Publication Title
A retinoblastoma allele that is mutated at its common E2F interaction site inhibits cell proliferation in gene-targeted mice.
Sample Metadata Fields
Specimen part
View Samples- Mus musculus
- 8 Downloadable Samples
- Affymetrix Mouse Gene 1.0 ST Array (mogene10st)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Autophagy maintains the metabolism and function of young and old stem cells.
Sample Metadata Fields
Specimen part
View Samples- Saccharomyces cerevisiae
- 7 Downloadable Samples
- Illumina HiSeq 2000, Illumina Genome Analyzer II
Description
Purpose: The goals of this study were to determine whether the spliceosome interacts with non-intronic mRNAs Methods: RNAseq was performed on RNA that immunoprecipitated with the yeast SMD1 protein. Tandem-affinity-purified RNAs were extracted and RNAseq libraries were generated using the EpiCentre ScriptSeq kit (v1). We also performed RNAseq experiments on rRNA depleted total RNA extracted from an exosome mutant (rrp6?), a temperature-sensitive splicing mutant (prp40-1) and a parental strain (BY4741). The rRNA was depleted using the Invitrogen RiboMinus kit, according to manufactureres procedures. The depleted RNA was subsequently treated with Turbo DNAse I (Ambion) and RNAseq libraries were generated using the EpiCentre ScriptSeq kit (v1). Results: The SM RNAseq data identified a number of non-intronic mRNAs that appeard to be bound by the spliceosome. Among these was the BDF2 mRNA, which enocdes for a bromo-domain protein. BDF2 was highly enriched in both SM-IP datasets and was therefore analyzed in more detail. To determine if other non-intronic mRNAs could be regulated by the spliceosome, we analysed the transcriptome in the rrp6?, the prp40-1 and a parental strain. Bioinformatic analysis of these data sets revealed that roughly 1% of the non-intronic mRNAs in yeast could be targeted by the spliceosome. TopHat revealed cannonical splice junctions in roughly 30 non-intronic mRNAs, indicating that these messages are spliced. Conclusions: We demonstrate, for the first time, that the spliceosome can regulate expression of non-intronic mRNAs via one and/or two RNA cleavage events. We refer to this process as Spliceosome Mediated Decay (SMD). Overall design: We report RNAseq data for two SM immunoprecipitation experiments and RNAseq datasets for the parental strain (BY4741), the prp40-1 mutant, and the rrp6? strain.
Publication Title
Spliceosome-mediated decay (SMD) regulates expression of nonintronic genes in budding yeast.
Sample Metadata Fields
Subject
View Samples- Mus musculus
- 6 Downloadable Samples
- Affymetrix Mouse Gene 1.0 ST Array (mogene10st)
Description
Autophagy is critical for protecting HSCs from metabolic stress. Here, we used a genetic approach to inactivate autophagy in adult HSCs by deleting the Atg12 gene. We show that loss of autophagy causes accumulation of mitochondria and an oxidative phosphorylation (OXPHOS)-activated metabolic state, which drives accelerated myeloid differentiation likely through epigenetic deregulations rather than transcriptional changes, and impairs HSC self-renewal activity and regenerative potential.
Publication Title
Autophagy maintains the metabolism and function of young and old stem cells.
Sample Metadata Fields
Specimen part
View Samples