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  • Pancreas and spleen were microdissected from wild type (C57B6/SV129 background) E14.5 embryos and P0 newborn pups. Pools of 30-60 tissues were used. Tissues were digested for 1 hour at 37°C in HBSS/0.1% collagenase A/20 µg/ml DNase I, followed by dissociation of the cell clusters into single cells using a non-enzymatic dissociation medium (Sigma). Single cell suspensions were blocked with mouse IgGs and anti-CD16/32 Abs and immunostained with biotin-anti-CD11b (Biolegend) and RPE-conjugated goat anti-CCR2 (R&D Systems), followed by Cy5.5 conjugated streptavidin. CD11b+CCR2+ cell were then isolated by FACS sorting. mRNA from purified cells was prepared using the RNAeasy kit (Qiagen) and run on a MouseWG-6 v2.0 Expression BeadChip.
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  • In this experiment the transcriptome reprogramming in barley during host and nonhost interaction with Magnaporthe sp. was analyzed in a time-series approach. Seven days old barley plants of cv. Vada were mock-inoculated or inoculated with adapted Magnaporthe isolate TH6772 from rice or non-adapted isolate CD180 from Pennisetum. After 6, 12, 24 and 48 hours the abaxial epidermis was sampled. Total RNA was extracted using the RNeasy Plant Mini Kit with on-column DNase digestion (Qiagen, Hilden, Germany), and hybridized to Agilent 44k oligonucleotide arrays.
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  • In this experiment the transcriptome reprogramming in barley during host and nonhost interaction with Puccinia sp. was analyzed in a time-series approach. Ten days old barley plants of cv. Vada were mock-inoculated or inoculated with P. hordei (Ph), 1.2.1 isolate, or P. triticina (Pt), BRW96258 isolate. After 12, 24, 36 and 48 hours first leaves were sampled. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), the Ambion TURBO DNA-free DNase Kit was used for DNA elimination, and RNA was hybridized to Agilent 44k oligonucleotide arrays.
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  • Hydrostatic pressure is one of the main mechanical stimuli cartilage cells are submitted to during joint loading. If moderate hydrostatic pressure is known to be beneficial to cartilage differentiation, excessive pressure, on the other hand, induces changes in cartilage similar to those observed in osteoarthritic cartilage. Therefore, the purpose of the experiment is to identify new target genes of high hydrostatic pressure in chondrocyte precursor cells.
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  • Gastrulation represents a pivotal point in mammalian development, when the basic body plan is established and cells are specified into one of the three germ layers. This is followed by rapid diversification into specific lineages and the appearance of the various cell types required to build each of the organs. The rich variety of cell types present at this stage has never been rigorously characterised in any mammalian organism, and thus insight into cell fate decisions and the underlying regulatory networks have been inaccessible. We have used droplet based single-cell RNA-sequencing to address this by profiling ~20000 cells from C57BL/6 E8.25 mouse embryos.
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  • Breast cancer was one of the first cancer types where molecular subtyping led to explanation of interpersonal heterogeneity and resulted in improvement of treatment regimen. Several multigene classifiers have been developed and in particular those defining molecular signatures of early breast cancers possess significant prognostic information. Hence since 2014, molecular subtyping of primary breast cancers was implemented as a part of routine diagnostics with direct impact of therapy assignment. In this study, we evaluate direct and potential benefits of molecular subtyping in low-risk breast cancers as well as present the advantages of a robust molecular signature in regard to patient work-up among high-risk breast cancers.
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  • RNA extraction and microarray analysis total RNA from immortalized normal mammary epithelial cells (184A1, MCF-12A), breast cancer cells (MDA-MB-231, MCF-7, MDA-MB-468, SK-BR-3), BCSC (MDA-MB-231SC, MCF-7SC, XM322, XM607). MDA-MB-231SC and MCF-7SC originating from breast cancer cell lines; XM322 and XM607 derived from clinical specimens which had been described in previous submission (E-MTAB-5057). The miRNA profiling was performed using Agilent miRNA array. Microarray experiments were conducted according to the manufacturer's instructions. To select the differentially expressed genes, we used threshold values of ≥ 2 and ≤ −2-fold change and a Benjamini-Hochberg corrected p value of 0.05. The data was Log2 transformed and median centered by genes using the Adjust Data function of Cluster 3.0 software then further analyzed with hierarchical clustering with average linkage (genes which value more than 100 were evaluated).
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  • Alzheimer’s disease (AD) is an aging related neurodegenerative pathology that affects millions of people worldwide. This pathology has been related to a range of features such as deposition of proteins in the brain, alterations in cell cycle, accumulation of DNA damage, DNA repair deficiency and mitochondrial dysfunction, however the molecular mechanisms implicated in its pathogenesis are still uncertain. Thereby, the present study aimed to evaluate whether peripheral blood mononuclear cells (PBMCs) of AD patients display alterations in gene expression profilescompared to age-matched controls. Blood samples were collected from 25 AD patients and 15 age-matched controls in order to perform genome-wide mRNA expression (microarray). In blood cells, Rank products bioinformatics analysis indicated 593 mRNAs adifferentially expressed in AD compared to controls. Our results demonstrated that PBMCs have alterations in cell cycle and DNA repair as proposed for AD neurons, reinforcing the idea that AD blood cells can provide information about AD status and also can be import as source of studies to better understand the disease.
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  • Somitogenesis is the segmentation of the developing embryonic body axis into somites and is guided by oscillating genes, which create waves of expression that travel across the presomitic mesoderm (PSM) from posterior to anterior. Upon arrival of a wave at the PSM's anterior end, a new somite is formed. To identify genes that are expressed in a wave-like pattern we dissected the PSM of four different mouse embryos (pre-turned), separated the left and right sides, and divided each into five segments, from posterior to anterior (sampling sites 1 to 5). Each segment was used to construct libraries for high-throughput RNA-sequencing. For one embryo, we also sequenced two somites.
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  • Gastrulation represents a pivotal point in mammalian development, when the basic body plan is established and cells are specified into one of the three germ layers. This is followed by rapid diversification into specific lineages and the appearance of the various cell types required to build each of the organs. The rich variety of cell types present at this stage has never been rigorously characterised in any mammalian organism, and thus insight into cell fate decisions and the underlying regulatory networks have been inaccessible. We have used droplet based single-cell RNA-sequencing to address this by profiling ~7000 cells from three E8.25 mouse embryos.
    Data Types:
    • Text
    • File Set
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