Project description:This dataset contains 48 RNA-seq samples from Triticum aestivum cv. Paragon plants grown under field conditions as part of the WGIN 2016 diversity trial. Flag leaf node tissues were harvested at eight time points following anthesis to capture transcriptional changes associated with senescence progression. Plants were subjected to two nitrogen fertilisation treatments (100 and 200 kg N ha⁻¹), with three biological replicates per treatment and time point. The selected time points for each nitrogen level were: N100 – 0, 7, 14, 21, 25, 28, 31, and 34 days post-anthesis (DPA); N200 – 0, 7, 14, 21, 28, 32, 35, and 38 DPA. This time-course RNA-seq dataset enables the investigation of gene expression dynamics during post-anthesis senescence in wheat flag leaf nodes, which serve as central hubs for nutrient trafficking during grain filling.
Project description:The tumour microenvironment is a critical element involved in tumour progression and responsiveness to therapies. Using functionalized tunable stiffness hydrogel, mimicking the mechanical properties of healthy and tumour tissues, we explore how the stiffness of the microenvironment can influence cancer cells by generating RNA-seq transcriptional profiles of 4T1 mouse breast cancer cells cultured on soft vs stiff polyacrylamide hydrogels for 24 hours.
Project description:Insulin binds the insulin receptor (IR), which in turn has showed to form nanoclusters at the cell membrane. Trying to exploit the nanoscale spatial organization of IR, we developed rod-like insulin-DNA-origami nanostructures carrying different numbers of insulin molecules. These structures (referred to as NanoRods, or NR) were then utilized to investigate receptor activity to spatial distribution of insulin molecules. One part of this investigation was to study the transcriptional response of brown adipocytes treated with NR bound to one insulin (NR-1) and NR bound to seven insulin (NR-7), and compare to untreated controls. Furthermore, free insulin was also used to ensure that similar pathways were activated when using NR-bound insulin.
Project description:In our study, we examined tissue samples from 20 cattle that had been experimentally infected with the FMDV strain O/FRA/1/2001. In 18 of these animals, the infection persisted for more than 28 days. At necropsy, epithelial tissue was collected from the dorsal nasopharynx and dorsal soft palate, the primary sites of persistent infection. Five biological replicates were taken from each location in each animal and analyzed using FMDV-specific RT-qPCR. A subset of these samples was then selected for transcriptome sequencing. Samples were disintegrated using a CP02 cryoPREP (Covaris) and mixed with AL buffer (Qiagen) and TRIzol LS (Invitrogen). After biocontainment removal, trichloromethane was added, and the mixture was centrifuged. About 400 µl of the RNA-containing upper aqueous phase was extracted using the Agencourt RNAdvance Tissue Kit (Beckman Coulter) with a KingFisher Flex processor (Thermo Fisher Scientific). RNA quantity and quality were measured with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). For transcriptomic analysis, 52 samples were selected, including 25 DNP and 23 DSP samples from 14 persistently infected animals and two that cleared the infection before 21 dpc. mRNA was isolated using the Dynabeads mRNA DIRECT Micro Purification Kit (Invitrogen), and libraries were prepared with the Colibri Stranded RNA Library Prep Kit (Invitrogen). ERCC internal control was added before mRNA extraction. The isolated mRNA was fragmented to ~150 nucleotides, adapters were ligated, and the RNA was transcribed into cDNA, which was amplified and indexed for Illumina libraries. The libraries' length and quality were assessed with an Agilent 4150 TapeStation and quantified using Qubit 2.0. Sequencing was performed on a NovaSeq machine in 100 bp single-end mode.
Project description:RELEVANCE: Smith-Lemli-Opitz syndrome (SLOS) is a human disease caused by mutations in the gene coding for the enzyme DHCR7 (7-dehydrocholesterol (7DHC) reductase), which catalyzes the final step of cholesterol biosynthesis. Accumulated 7DHC in tissues and body fluids of SLOS patients gives rise to numerous oxidation products (oxysterols) in situ, some of which are cytotoxic, and which may contribute to the pathophysiology of SLOS. The SLOS phenotype is broad, ranging from death in utero to viable individuals with malformations and malfunctions in numerous organ systems and tissues, including neurological and cognitive defects. The latter presentations suggest that impaired cholesterol synthesis, in particular the generation of toxic oxysterols, has a deleterious impact on the morphogenesis and viability of neurons in the CNS. In a rat model of SLOS (using a small molecule inhibitor of DHCR7), the loss of photoreceptors was also documented, distinct from the continuous viability of other retinal neurons and supporting cells, and this selective cell death was recapitulated in vitro using 661W cells (an immortalized line derived from mouse cones) incubated with purified 7DHC-derived, SLOS-associated oxysterols. Upon exposure to these compounds, cell viability assay results for 661W showed one to two orders of magnitude higher sensitivity with respect to efficacy and potency, and also an accelerated time frame for cell death, compared to retinal Mueller glia and retinal pigment epithelial cells. These findings inspired questions as to the molecular mechanisms underlying oxysterol-induced neuronal cell death. Therefore, we characterized differential gene expression associated with processes and pathways induced by oxysterol treatments, first, to provide insights regarding cell death and dysfunction, not only in SLOS, but also extended to other neurodegenerative diseases (including those affecting the retina), and second, to identify cellular protective responses, with the expectation that these findings would suggest possibilities for future prevention and treatment of neurological disease and damage. INTENT: Generate gene expression array profiles of 661W cells following exposure to either of two oxysterols. The oxysterols are employed at doses already determined to exert full cytotoxicity by 24 hours incubation time; however, for the purposes of the array, stop incubations for each oxysterol at time points preceding global cell death, to harvest still-intact RNA from a majority of cells that have maintained membrane integrity, and are also displaying microscopically observable indications of morphological response to the oxysterol treatments already known to precede the demise of the cells. Therefore, the samples are expected to manifest transcriptomes emblematic of gene expression changes in response to the oxysterols. These changes should fit patterns that correlate with pathways and processes associated with cellular damage and regulated cell death, or with cell survival/protective and repair mechanisms (with expected overlap of the two opposing scenarios). As a negative control, a separate set of replicates are incubated with cholesterol under conditions having no impact on cell viability (23 hours, at a (non-physiological) concentration intermediate between that of the two oxysterols). To identify differentially expressed genes, individual array probe set data for either oxysterol and for cholesterol are matched with those from cells incubated with a vehicle control (for 24 hours), for computing “-fold change” in expression and statistical significance of expression differences. EXPERIMENTAL WORKFLOW: 1) 661W cells were seeded in 100-mm cell culture-treated dishes at a density permitting proliferation to subconfluence, allowing cells to retain neurite-like extensions; includes overnight adaptation to a simplified incubation medium devoid of most growth factors and reagents supporting antioxidant activity. 2) Cells were exposed for a predetermined time period to a single concentration of: i) either of two different oxysterols (EPCD, an endoperoxide specific to SLOS, or 7-ketocholesterol); ii) cholesterol; or iii) vehicle control. 3) Total RNA was harvested, from each triplicate sample representing the above treatments, according to the RNeasy Plus minikit protocol (Qiagen). 4) Final sample preparation (amplification, labeling, fragmentation of cRNA) was carried out a core facility following Affymetrix specifications and protocols. 5) Hybridized chips were scanned to generate raw intensity data for further analysis.
Project description:We generated RNA-seq data of 30 cases of acute leukemia of ambiguous lineage (ALAL), including both bilineal and biphenotypic patients, in order to develop methods to improve the diagnosis of this disease. First, we used an in-house pipeline to comprehensively detect genetic lesions in RNA-sequencing data. Second, we generated a machine learning (ML) classifier trained on compared ALAL gene expression profiles (GEPs) with representative AML (n=145), B-ALL (n=223) and T-ALL (n=85) cases.
Project description:Total RNA-seq of blasts derived 100 adult T-ALL cases, 211 AML cases and 13 mixed myeloid/lymphoid leukemias with CpG Island Methylator Phenotype (CIMP). In addition, CD34+ HSPCs derived from 9 healthy donors are used as a control. Due to patient confidentiality considerations, the raw data files for this dataset have been deposited to the EGA controlled-access archive under the accession numbers EGAS00001007094 (study); EGAD00001011054, EGAD00001007646, EGAD00001007581 (datasets).
Project description:Male C3H/HeOuJ and CAST/EiJ mice were treated with diethylnitrosamine to induce liver tumours. We collected liver samples from untreated P15 mice for control experiments. Liver tissue samples were snap frozen in liquid nitrogen and total RNA was extracted using the AllPrep 96 DNA/RNA Kit (Qiagen) according to the manufacturer’s instructions. Libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold (Illumina) and sequenced on an Illumna HiSeq4000 to produce 150bp paired-end reads.
Project description:Esophageal squamous cell carcinoma (ESCC) is among the most common malignancies, but little is known about the spatial intratumor heterogeneity (ITH) and the temporal clonal evolutionary processes in this cancer. Interestingly, the epigenetic profiling also showed strong evidence of spatial ITH, and the phyloepigenetic trees were extremely similar with the phylogenetic ones, indicating the interplay and co-dependency of genetic and epigenetic alterations in ESCC. We found that several genes were both mutated and hypermethylated at their promoters, such as ASXL1 and EPHA7. Our integrated investigations of the spatial ITH and the temporal clonal evolution might provide insights into developing biomarkers for early diagnosis of ESCC, as well as personalized therapeutic targets for treating this malignancy. DNA methylation profiles of 12 tumor regions and 2 matched normal esophageal epithelial tissues from three M-WES-examined ESCC cases (ESCC01, ESCC03 and ESCC05) were performed using Illumina Infinium HumanMethylation450K platform (Illumina, San Diego, CA) at the Epigenome Center of University of Southern California.