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We performed a large-scale expression analysis with inducible gain-of-function (GOF) lines which contain a C-terminal fusion protein of the TF of interest and a glucocorticoid receptor (GR) domain, driven by a constitutive 35S promoter. Such fusion proteins reside in the cytosol and can only translocate to the nucleus in the presence of dexamethasone (DEX), enabling the TF to regulate its downstream target genes (Corrado and Karali, 2009). In total 18 GOF lines were used of the following TFs: ERF-1 (AT4G17500), ERF2 (AT5G47220), ERF5 (AT5G47230), ERF6 (AT4G17490), ERF11 (AT1G28370), ERF8 (AT1G53170), ERF9 (AT5G44210), ERF59 (AT1G06160), ERF98 (AT3G23230), MYB51 (AT1G18570), STZ (AT1G27730), WRKY28 (AT4G18170), WRKY33 (AT2G38470), WRKY15 (AT2G23320), ZAT6 (AT5G04340), WRKY48 (AT5G49520), RAP2.6L (AT5G13330) and the control 35S::GFP-GR. To get an indication of which genes are direct or indirect targets of the induced TF, we performed a time-course experiment. All GOF lines were transferred at 15 DAS to DEX-containing medium and the third leaf was harvested at 1 h, 2 h, 4 h, 8 h and 24 h after transfer. The expression of 30 TFs (17 TFs listed above and WRKY6 (AT1G62300), WRKY30 (AT5G24110) and WRKY40 (AT1G80840), GA2OX6 (AT1G02400), GA20OX1 (AT4G25420), ACS6 (AT4G11280), MPK3 (AT3G45640), DEL1 (AT3G48160), UVI4 (AT2G42260), CYCA3;1 (AT5G43080), CYCB1;2 (AT5G06150), XET (AT1G10550), EXPA5 (AT3G29030)) was measured with nCounter Nanostring (Geiss et al, 2008). The time-course experiment gives an indication of whether a gene is putatively a direct, and thus induced during the early time points, or an indirect target of the induced TF.
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In order to gain a global temporal picture of epiblast potency across implantation, we collected mouse embryos at E4.5, E4.75 and E5.0, and dissected the epiblast for deep-sequencing analysis.
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Myocyte enhancer factor 2 contributes as a transcription factor to cardiac remodeling processes. We wanted to link and compare known myocyte enhancer factor 2 targets to overall targets. Therefore, we used a model of neonatal rat cardiomyocytes and precipitated sheared chromatin samples with 5µg of MEF2 Ab.
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Natural Killer Gene Complex (NKC)-encoded immunomodulatory C-type lectin-like receptors (CTLR) include members of the NKRP1 and CLEC2 gene families, which constitute genetically linked receptor-ligand pairs and are thought to allow for NK cell-mediated immunosurveillance of stressed or infected tissues. The mouse CTLR Nkrp1g was previously shown to form several receptor-ligands pairs with the CLEC2 proteins Clr-d, Clr-f, and Clr-g, respectively. Recently, we demonstrated a gut-restricted expression of Clr-f on intestinal epithelial cells that is spatially matched by Nkrp1g on subsets of intraepithelial lymphocytes (Leibelt et al., 2015). We now investigated expression and ligand interaction of Nkrp1g in the splenic compartment, and found an exclusive expression of Nkrp1g on a small subset of NK cells that upregulates Nkrp1g after cytokine exposure. To further characterize NKrp1g+ NK cells, we performed microarray analysis of resting Nkrp1g+ versus Nkrp1g- splenic NK cells that were purified by FACsorting. Total RNA isolation was followed by an Affymetrix microarray analysis.
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We characterized single-cell transcriptional profiles of the cardiac non-myocyte cell pool in C57BL/6J mice. The cell preparation we sequenced consisted of metabolically active, nucleated non-myocyte cells from heart ventricles of female and male mice which were depleted of endothelial cells. The goals of this experiment included examining cellular diversity, identifying markers of understudied cell populations, exploring functional roles of different cell types, and characterizing sexual dimorphism in cardiac gene expression.
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We aimed to study how production of p-coumaric acid, a precursor of multiple secondary aromatic metabolites, influences the cellular metabolism of Saccharomyces cerevisiae. We evaluated the growth and p-coumaric acid production in batch and chemostat cultivations and analyzed the transcriptome and intracellular metabolome during steady state in low- and high-producers of p-coumaric acid in two strain backgrounds, S288c or CEN.PK. For analysis of the differential gene expression, we did pairwise comparisons between the optimized and non-optimized strains for p-CA production: CEN.PK strains (ST4288 and ST4408) and the S288c strains (ST4353 and ST4397). Transcriptome analysis showed that the CEN.PK strain was less affected by engineering towards higher p-CA production than the S288c strain, as the number of significantly up-/down-regulated genes was correspondingly 652 and 1927 amongst others, strain S288c had downregulations in gene sets involved in amino acid and protein biosynthesis. This suggests that CEN.PK may be a better platform strain for production of aromatic compounds than the S288c strain.
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RNA-sequencing of twelve treatments: two cultivars (Embrapa 45 and BR 4), two oxygen conditions [fully aerobic state (normoxy) and hypoxic], and three treatment sampling times (0.5h, 4h, and 28h). For each of twelve treatments, equimolar quantities of purified total RNA from roots of twelve plants [three biological replicates (four plantlets per replicate)] were pooled to result one library. After processing the twelve libraries (poly-A purification, fractionation, cDNA synthesis using random primers, and ligation to bar-coded adapters), fragments of 150250 pb were isolated and multiplexed, resulting one sequencing library (a pooled of equimolar quantities from twelve initial libraries; each library with a specific barcode for further bioinformatic discrimination). Sequencing library was used to produce clusters for a 1 x 100 bp single end-sequencing run into one lane on a flow cell for sequencing in a Hi-Seq 2000 (Illumina).
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This experiment was designed to compare transcription profile between healthy controls and patients of rheumatoid arthritis.
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We have discovered that neutrophils that infiltrate the spinal cord of mice with EAE, a model of multiple sclerosis, but not intravascular neutrophils that crawl on the luminal endothelial surface, bear on their surface the adhesion molecule Icam1. The goal of this experiment was to use Icam1 to isolate these two neutrophil subpopulations in order to compare their transcriptomes and gain insights into their properties. To this end, EAE was induced in C57BL/6J mice by immunization with myelin oligodendrocyte glycoprotein (MOG) peptide (a.a. 35-55) and adjuvants (i.e. complete Freund’s adjuvant and pertussis toxin). On day 15 post-immunization, intravascular neutrophils (CD45hiCD11b+CD11c−Ly6g+Icam1−) and extravasated neutrophils (CD45hiCD11b+CD11c−Ly6g+Icam1+) were isolated from spinal cords by FACS. For comparison, we simultaneously isolated two other populations of myeloid cells: macrophages (CD45hiCD11b+CD11c−Ly6g−) and dendritic cells (CD45hiCD11b+CD11c+Ly6g−). RNA was analyzed in biological duplicate using Affymetrix GeneChip Mouse Gene 2.0 ST arrays.
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Here, we profiled transcription across the cell cycle in single human myxoid liposarcoma cells, without prior synchronization. Flourescence-activated cell sorting was used to infer G1, S or G2/M cell cycle stages after staining cells for DNA content. Single-cell RNA-sequencing was performed according to the Smart-seq2 protocol.
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