ChIP-seq of definitive endoderm cells derived in vitro from human embryonic stem cells in order to study the role of transcription factor GATA6 in human pancreas development
ABSTRACT: Mutant GATA6 hPSCs were obtained by TALEN genome editing or re-programmed from patient fibroblasts. Along with wild-type H9 cells, these GATA6 mutant cell lines were differentiated into SOX17+/CXCR4+ endodermal cells (day 3). The purpose of this work was to study the role of GATA6 in the development of the human pancreas at a molecular level.
Project description:In this study, we generate genomic maps of Mediator, Pol II, TBP, TFIIH, TFIIA, TFIIB, TFIIE, TFIIF, by ChIP coupled to next generation sequencing technology (ChIP-seq), in wild type strains from Saccharomyces cerevisiae and in a mutant for the Mediator essential subunit Med10
Project description:Oxidative Stress Protection and the Repair Response To Hydrogen Peroxide in the Hyperthermophilic Archaeon Pyrococcus furiosus Pyrococcus furiosus is a shallow marine, anaerobic archaeon that grows optimally at 100°C. Addition of H2O2 (0.5 mM) to a growing culture resulted in cessation of growth with a 2 hour lag before normal growth resumed. Whole genome transcriptional profiling revealed that the main response occurs within 30 min of peroxide addition, with the up-regulation of 62 open reading frames (ORFs), 36 of which are part of 10 potential operons. More than half of the up-regulated ORFs are of unknown function while some others encode proteins that are involved potentially in sequestering iron and sulfide, in DNA repair and in generating NADPH. This response is thought to involve primarily damage repair rather than protection, since cultures exposed to sub-toxic levels of H2O2 were not more resistant to the subsequent addition of H2O2 (0.5 – 5.0 mM). Consequently, there is little if any induced protective response to peroxide, rather, the organism maintains a constitutive protective mechanism involving high levels of oxidoreductase-type enzymes such as superoxide reductase, rubrerythrin and alkyl hydroperoxide reductase I. The related hyperthermophiles P. woesei and Thermococcus kodakaraensis were more sensitive to peroxide than P. furiosus, apparently due to the lack of several of its peroxide-responsive ORFs. Pyrococcus furiosus (DSM 3638) was grown at 95°C in a 20-liter fermentor using maltose as the carbon and energy source. An exponential-phase culture of P. furiosus that had undergone three successive transfers in the experimental medium was used to inoculate the 20-liter fermentor. The culture was shocked with 0.5 mM of hydrogen peroxide (H2O2) when cell density was in mid-exponential phase (~ 5.0 ´ 107 cells/ml, as determined by direct microscopic cell count). To obtain RNA for microarray and for quantitative PCR (QPCR) analyses, samples (2 liter) were rapidly removed from the fermentor and cooled to 4°C. Total RNA was extracted using acid-phenol and stored at -80°C until needed. A total of 3 biological replicates in triplicate (3 copies on the same slide) was used in the data set.
Project description:High-resolution genome-wide mapping of the yeast transcription machinery. ChIP-chip was performed to identify the genomic binding locations for each factor. <br><br>Processed data files are also available on the FTP server for this experiment.
Project description:The aim of the experiment is to identify genome wide binding sites for the transcription factor MYCN in MYCN non-amplified and MYCN amplified human neuroblastoma cell lines. Datasets are presented for the ChIP-seq analysis in the tetracycline inducible cell line SH-SY5Y-MYCN (SH-SY5Y/6TR(EU)/pTrex-Dest-30/MYCN), derivative of the parental cell line SH-SY5Y; for noninduced cells and for 24 and 48 hours of Tet induction. Analysis for patinet matched MYCN amplified cell lines SMS-KCN and SMS-KCNR is also included.
Project description:In angiosperms, the endosperm provides nutrients for embryogenesis or seed germination and is the primary tissue where gene imprinting occurs. To map the imprintome of the early developing endosperm in maize, we performed high-throughput transcriptome sequencing of the kernels at 0, 3, 5 days after pollination (DAP) and the endosperms at 7, 10, and 15 DAP produced from the B73 and Mo17 reciprocal crosses. We observed a gradual increase of paternal gene mRNAs in the 3- and 5-DAP kernels. In the 7-DAP endosperm, the majority of the tested genes reached a ratio of 2m:1p suggesting that paternal genes were nearly fully activated by 7 DAP. A total of 116, 234 and 63 imprinted genes exhibiting parent-specific expression were identified in the 7-, 10-, and 15-DAP endosperms, respectively. The highest amount of paternally expressed genes (PEGs) was found at 7 DAP mainly due to the significantly deviated parental allele expression ratio of these genes at this stage, while nearly 80% of the maternally expressed genes (MEGs) were specific to 10 DAP which were primarily attributed to the sharply increased expression levels compared to the other stages. GO enrichment analysis of the imprinted genes indicated the 10-DAP-specific MEGs were involved in the nutrient uptake and allocation through auxin signaling pathway in the maize endosperm coincident with the endosperm developmental stage associated with the beginning of starch and storage protein accumulation The unpollinated kernels (0 DAP), the kernels of 3, 5 DAP and endosperms of 7 10, 15 DAP from the B73 and Mo17 reciprocal crosses were used to perform high-throughput sequencing using the Illumina HiSeq2000 platform