RNA-Seq of mouse embryonic stem cells derived from genetically diverse inbred mouse strains
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ABSTRACT: We measured genome-wide gene expression of embryonic stem cells derived from eight genetically diverse inbred mouse strains. We derived and cultured cells in LIF+2i media with feeders, and removed feeders prior to RNA collection. All lines were passage 6-8 when RNA was collected. We collected RNA from three biological replicate cell lines derived from each of the eight inbred mouse strains. For three strains (C57BL/6J, NOD, and PWD/PhJ), we obtained technical replicate RNA-Seq runs of each of the three biological replicate cell lines.
Project description:Salmonella Typhimurium ST313 is associated with invasive nontyphoidal salmonellosis (iNTS) disease in sub-Saharan Africa. Using ST313 isolate, D23580, and the well-characterized ST19 isolate 4/74, that causes gastroenteritis across Europe, we generated transcriptomic data from sixteen infection-relevant in vitro conditions and during infection of murine macrophages (http://bioinf.gen.tcd.ie/cgi-bin/jay/salcom_v2.pl?db=salcom_africa_new_HL). The transcriptional response of the two S. Typhimurium pathovariants to environmental stress was broadly similar. Comparative gene expression analysis identified virulence and metabolic genes that were up/down-regulated in D23580 versus 4/74. Findings were backed up by proteomic analysis, with12 genes being differentially-expressed at both the transcriptional and protein levels including melA and cysS. The data led us to discover that the ablation of melibiose utilization was caused by 3 SNP mutations in D23580 and to identify a novel plasmid-maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase. Our functional transcriptomic analysis represents a powerful tool for identification of key phenotypic differences between bacterial pathovariants.
Project description:The cell wall is among the first plant structures encountered by necrotrophic fungal pathogens, such as Botrytis cinerea. The composition of plant cell walls varies depending on the species, type of cell or tissue, and stage of development. Cell walls are important reservoirs of energy-rich sugars for pathogens, but also are barriers that impair colonization of host tissues. Growing fungal hyphae secrete enzymes that hydrolyze cell wall polysaccharides. Degradation of wall polysaccharides provides nutrients for the pathogen and improves the access of secreted Botrytis enzymes to all host cell wall targets and cytoplasmic constituents. Destruction of host cell walls results in tissue maceration, a hallmark of diseases caused by Botrytis. The Botrytis genome encodes 1,155 predicted carbohydrate-active enzyme (CAZy) genes; products of 275 are potentially secreted. Transcriptome sequencing identified Botrytis CAZy genes expressed during infections of lettuce leaves, ripe tomato fruit and grape berries. On all three hosts, Botrytis expresses a common group of 229 predicted CAZy genes including 28 pectin-modifying enzymes, 21 hemicellulose-modifying proteins, 18 enzymes targeting pectin and hemicellulose side-branches, and 16 enzymes that may degrade cellulose. Pectin polysaccharides are abundant in grape and tomato cell walls, but lettuce leaf walls are predominantly hemicelluloses and cellulose. These results suggest that Botrytis targets similar wall polysaccharide networks; e.g., pectins, on leaves and fruit, but also attacks unique host wall polysaccharide substrates The diversity of the Botrytis CAZy proteins may be partly responsible for its wide host range. 3 biological replicates consisting of groups of infected tomato fruits from different plants
Project description:Serum-to-2i interconversion of mouse Embryonic Stem Cells (mESCs) is a valuable in vitro model for early embryonic development. To assess whether 3D chromatin organization changes during this transition, we established Capture Hi-C with target-sequence enrichment of DNase I hypersensitive sites. We detected extremely long-range intra- and inter-chromosomal interactions between a small subset of H3K27me3 marked bivalent promoters involving the Hox clusters in serum grown cells. Notably, these promoter-mediated interactions are not present in 2i ground-state pluripotent mESCs but appear upon further development into primed-like serum mESCs. Reverting serum mESCs to ground-state 2i mESCs removes these promoter-promoter interactions in a spatiotemporal manner. H3K27me3, which is largely absent at bivalent promoters in ground-state 2i mESCs, is necessary but not sufficient to establish these interactions, as confirmed by Capture Hi-C on Eed-/- serum mESCs. Our results implicate H3K27me3 and PRC2 as critical players in chromatin alteration during priming of ESCs for differentiation. To study dynamics in chromatin architecture and to characterize long-range interaction, we performed Hi-C using DpnII as the restriction enzyme, potentially reaching a genome-wide coverage at a less than 1Kb resolution. We subsequently performed enrichment of interaction by a target capture similar to the exome sequencing approach. We enriched for DNaseI hyper-sensitive sites (DHSâs) in chromatin from mESCs. Probes were designed against the union of all DHSâs of Serum and 2i mESCs. Capture Hi-C reveals Extremely Long-Range Interactions (ELRI) in Serum but not in 2i ESCs. We observed H3K27me3 as a prominent characteristic, but not exclusive feature of ELRI loci in Serum mESCs. To further elucidate the involvement of constituents of PRC1 and PRC2 in ELRI, we performed ChIP-seq experiment on Suz12 and Ring1B during serum-to-2i transition. In addition, RNA-seq was performed to compare the expression levels of genes.
Project description:Selective maintenance of genomic methylation imprints during pre-implantation development is required for parental origin-specific expression of imprinted genes. The Kruppel-like zinc finger protein ZFP57 acts as a factor necessary for maintaining the DNA methylation memory at multiple imprinting control regions (ICRs) in early mouse embryos and ES cells. Maternal-zygotic deletion of ZFP57 in mice presents a highly penetrant phenotype with no animals surviving to birth. In addition, several cases of human transient neonatal diabetes (TND) are associated with somatic mutations in ZFP57 coding sequence. Here we comprehensively map sequence-specific ZFP57 binding sites in an allele-specific manner using hybrid ES cell lines from reciprocal crosses between C57BL/6J and Cast/EiJ mice assigning allele specificity to approximately two thirds of all binding sites. While half of these are biallelic and include ERV targets, the rest show mono-allelic binding based either on parental-origin or on genetic background of the allele. Parental-origin allele-specific binding was methylation-dependent and mapped only to imprinted DMRs established in the germline (gDMRs). No binding was evident at secondary somatically-derived DMRs. ZFP57-bound gDMRs can predict imprinted gene expression and we identify new imprinted genes, including the Fkbp6 gene with a critical function in mouse male germ cell development. Genetic-background specific sequence differences also influence ZFP57 binding. We show that genetic variation that disrupts the consensus binding motif and its methylation is associated with mono-allelic expression of neighbouring genes. The work described here uncovers further roles for ZFP57 mediated regulation of genomic imprinting and identifies a novel mechanism for genetically determined mono-allelic gene expression. Input and Zfp57 CHiP-Seq profiles of hybrid Black6/Cast ES cells were generated by sequencing using the Illumina GAIIx platform.
Project description:Embryonic stem cells from B6 and NOD backgrounds were derived freshly in the presence of 2i. After 3-5 passages on feeders, ES cells were cultured in 2i media without any feeders for several passages. In order to identify differentially expressed genes and proteins, we performed RNA-Seq and mass spectromety analysis respectively. Among the differentially expressed genes, we identified several important players in innate and adaptive immunity. Several of these genes had been linked to onset of type-1 diabetes. Proteomics analysis was able to quantitative differences in protein expression among the B6 and NOD ES cell lines.
Project description:Purpose: The aim of this study was to profile the transcriptome of differentiating embryonic stem cells (ES) with nonsilencing shRNA mediated knockdown and Setdb1 geneTrap heterozygous cells with Setdb1 shRNA mediated knockdown. Methods: Female Setdb1+/+ XistâA/+ ES cells were induced to differentiate and RNA sampled on day 0, day 3 and day 5 of differentiation. RNAseq libraries were prepared with TruSeq RNA sample preparation v2 kit. Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads. Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data. Total RNA was extracted and purified from each cell type and their transcriptomes analysed by RNA-Seq.
Project description:In this study, a whole-genome CombiMatrix Custom oligonucleotide tiling microarray with 90000 probes covering six sequenced Helicobacter pylori(H. pylori) genomes was designed and utilized for comparative genomic profiling of eight unsequenced strains isolated from patients with different gastroduodenal diseases in Heilongjiang province of China. Since significant genomic variation were found among these strains, an additional 76 H. pylori stains with different clinical outcomes isolated from various provinces of China were further tested by PCR to demonstrate this distinction. We observed several highly variable regions among strains of gastritis, gastric ulceration and gastric cancer. They are involved in genes associated with bacterial type I, type II and type III R-M system as well as in a virB gene neighboring the well studied cag pathogenic island. Previous studies have reported the diverse genetic characterization of this pathogenic island, but it is conserved in the strains tested by microarray in this study. Moreover, a number of genes involved in the type IV secretion system related to DNA horizontal transfer between H. pylori strains were identified based on the comparative analysis of the strain specific genes. These findings may provide new insights for discovering biomarkers for prediction of gastric diseases. Here we describe the design and use of a high-density oligonucleotide microarray covering six sequenced H. pylori genomes as well as several sequenced plasmids. The performance of this microarray is evaluated, and its utility is illustrated for the hybridization of genomic DNA in order to compare eight uncharacterized H. pylori strains which have not been sequenced with the six known, sequenced strains. We utilize this microarray to identify variable genomic region among H. pylori strains isolated from patients with different gastroduodenal diseases in a Chinese patient population. H. pylori isolates from 2 patients with chronic superficial gastritis, 2 patients with atrophic gastritis, 2 patients with gastric ulcer, and 2 patients with gastric cancer were studied. All eight strains were isolated from Heilongjiang province of China. A number of variable regions with high genetic diversity was identified. 26 selected genes were validated by large scale PCR in both microarray tested strains while an additional 76 strains were isolated from eight provinces.
Project description:We show that aneuploidy is common in wild isolates of yeast, which are inherently tolerant to chromosome amplification and down-regulate expression at 40% of amplified genes. To dissect the mechanism of this dosage response, we generated isogenic strain panels in which diploid cells carried either two, three, or four copies of the affected chromosomes. Using a mixture of linear regression (MLR) model to classify genes, we find that expression is actively down regulated in proportion to increased gene copy at up to 30% of genes. Genes subject to dosage control are under higher expression constraint – but show elevated rates of gene amplification – in wild populations, suggesting that dosage compensation buffers copy number variation (CNV) at toxic genes RNA-seq and transcriptome analysis of S. cerevisiae natural isolates having aneuploidy. Technical triplicate was performed for isogenic diploid strains having 2, 3 and 4 copies of a given chromosome (strain panels), while technical duplicate or singulate was performed on all other aneuploids.
Project description:Mouse embryonic stem cells (mESCs), derived from pre-implantation blastocyst cells, can be maintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Many efforts have been devoted to identify genes that promote exit from the pluripotent state and the transition to a primed state of differentiation. One of the first identified players in this process was the Wnt/b-catenin effector TCF7L1 (previously referred to as TCF3), belonging to the family of four TCF/LEF transcription factors, which acts as pro-differentiation factor by repressing pluripotency genes. Of note, there is little evidence that the genetic abrogation of the mechanisms required for the exit from the pluripotent state is sufficient to enable self-renewal in the absence of 2iL. Here, we found that complete loss-of-function of Tcf7, Lef1, Tcf7l1 and Tcf7l2, the genes encoding for the four TCF/LEF transcription factors, (refered to as qKO) allows mESCs to become fully 2iL-independent and to propagate in basal N2B27. To understand the genetic program that allows qKO cells to achieve 2iL-independent self-renewal, we performed RNA sequencing (RNA-seq) of qKO and wild type mESCs.