Native Tn-seq analysis of insH3 in Escherichia coli
Ontology highlight
ABSTRACT: The experiment contains native Tn-seq data for Escherichia coli strain MG1655. The strain was grown at 37 degrees in LB medium and genomic DNA was isolated. We then used PCR to select for DNA regions containing a junction between insH3 and chromosomal DNA. Libraries were then prepared using these DNA fragments.
Project description:The experiment contains native Tn-seq data for Acinetobacter baumannii strain AB5075 with different genetic alterations. The strain was grown at 37 degrees in LB medium and genomic DNA was isolated. We then used PCR to select for DNA regions containing a junction between ISAba13 and chromosomal DNA. Libraries were then prepared using these DNA fragments.
Project description:Differential gene transcription enables development and homeostasis in all animals and is regulated by two major classes of distal cis-regulatory DNA elements (CREs), enhancers and silencers. While enhancers have been thoroughly characterized, the properties and mechansisms of silencers remain largely unknown. By an unbiased genome-wide functional screen in Drosophila melanogaster S2 cells, we discover a class of silencers that bind one of three transcription factors (TFs) and are generally not included in chromatin-defined CRE catalogs, as they mostly lack detectable DNA accessibility. The silencer-binding TF CG11247, which we term Saft, safeguards cell fate decisions in vivo and functions via a highly-conserved domain we term ZAC and the corepressor G9a, independently of G9a’s H3K9-methyltransferase activity. Overall, our identification of silencers with unexpected properties and mechanisms has important implications for the understanding and future study of repressive CREs, as well as the functional annotation of animal genomes.
Project description:In this study, we show that by simple modulation of extrinsic signaling pathways, a new class of pluripotent stem cells, referred to as region selective epiblast stem cells (rsEpiSCs), could be efficiently derived from different stages of the early embryo. rsEpiSCs share features of primed pluripotency yet are distinct from EpiSCs in their molecular characteristics and ability to colonize post-implantation embryos. We performed whole-genome bisulfite sequencing (WGBS) experiments to compare the DNA methylation landscapes of conventional EpiSCs and rsEpiSCs. Compare the DNA methylation profiles in 2 pluripotent stem cell types (LP-EpiSCs and conventional EpiSCs) in mouse. Two replicates are examined for each cell type.
Project description:Transposon insertion site sequencing (TIS) is a powerful method for associating genotype to phenotype. However, all TIS methods described to date use short nucleotide sequence reads which cannot uniquely determine the locations of transposon insertions within repeating genomic sequences where the repeat units are longer than the sequence read length. To overcome this limitation, we have developed a TIS method using Oxford Nanopore sequencing technology that generates and uses long nucleotide sequence reads; we have called this method LoRTIS (Long Read Transposon Insertion-site Sequencing). This experiment data contains sequence files generated using Nanopore and Illumina platforms. Biotin1308.fastq.gz and Biotin2508.fastq.gz are fastq files generated from nanopore technology. Rep1-Tn.fastq.gz and Rep1-Tn.fastq.gz are fastq files generated using Illumina platform. In this study, we have compared the efficiency of two methods in identification of transposon insertion sites.
Project description:We have demonstrated that a newly evolved TRS within the Nucleocapsid gene of SARS-CoV-2 (termed N.iORF3) leads to the expression of a novel subgenomic mRNA encoding a truncated C-terminal portion of Nucleocapsid, which is an antagonist of type I interferon production. Using reverse genetics-derived viruses we show N.iORF3 contributes to viral fitness during infection and observe distinct phenotypes when the Nucleocapsid coding sequence is mutated compared to when the TRS alone is ablated. Competition assays were performed to determine the relative fitness of different virus mutants and amplicons were analysed to quantify the proportions of different viruses in tissue culture.
Project description:Investigations of 5-hydroxymethylcytosine (5hmC) in biologically and clinically samples and models with low cell numbers have been hampered by the low sensitivity and reproducibility using current 5hmC mapping approaches. Here, we develop a selective 5hmC chemical labeling approach using tagmentation-based library preparation in order to profile nanogram levels of 5hmC isolated from ~1,000 cells (nano-hmC-Seal). Using this technology, we profiled the dynamics of 5hmC across different stages of mouse hematopoietic differentiation. Additionally, applying nano-hmC-Seal to the hematopoietic multipotent progenitor cells in an acute myeloid leukemia (AML) mouse model, we identified leukemia-specific, differentially hydroxymethylated regions that harbor previously reported and as-yet-unidentified functionally relevant factors. The change of 5hmC patterns in AML strongly correlates with the altered gene expression on a global scale. Together, our new approach offers a highly sensitive and robust method to study and detect DNA methylation dynamics from in vivo model and clinical samples. Selective 5hmC chemical labeling approach using tagmentation-based library preparation in order to profile nanogram levels of 5hmC isolated from ~1,000 cells
Project description:The biodegradation of lignocellulose requires the disruption of its lignin, which shields the metabolically assimilable polysaccharides in this recalcitrant natural composite. Although a variety of microorganisms can attack lignocellulose, white rot basidiomycetes are uniquely efficient at this process, cleaving the recalcitrant intermonomer linkages of lignin via extracellular oxidative mechanisms and mineralizing many of the resulting fragments to carbon dioxide via intracellular processes. Considerable progress has been made in understanding this process in the model white rot fungus Phanerochaete chrysosporium, which expresses important components of its ligninolytic system in response to nutrient limitation, as part of its secondary metabolism. Biochemical and genetic evidence point to an important role in P. chrysosporium for secreted lignin peroxidases (LiPs), manganese peroxidases (MnPs), and H2O2-producing oxidases, which are thought to work together to cleave lignin into low molecular weight fragments. However, many aspects of ligninolysis by P. chrysosporium remain poorly understood. Although a definitive picture of the entire ligninolytic system in P. chrysosporium is not yet attainable, transcriptome analyses of the fungus grown on wood can provide useful clues. With the advent of the initial genome assembly and annotations (v1.0 and v2.1), microarray-based transcriptome analysis allowed examination of transcript levels of P. chrysosporium genes when grown in ball-milled wood and in defined growth media. This approach provided useful insights but was limited to 10048 v2.1 targets and complicated by the unpredictable manner in which the fungus responds to unnatural carbon sources in submerged basal salts media. A complete, fully coordinated ligninolytic system is likely not expressed by P. chrysosporium on ball-milled wood, because a potential route for regulatory feedback has been eliminated: the cellulose and hemicellulose in this substrate is readily accessible to enzymes, and thus ligninolysis is not essential for growth. An alternative approach is to compare levels of gene expression just before and after the onset of secondary metabolism and extracellular substrate oxidation by P. chrysosporium as it utilizes solid wood as its carbon source. If this can be done, and decay of the substrate is also confirmed, then the genes undergoing marked changes in expression during the metabolic transition can be identified with greater confidence. Although not all such genes are expected to have roles in biodegradation, this strategy may identify interesting candidates for future investigation. Here we report RNAseq-based transcriptomes to characterize changes in gene expression that occur during the transition to ligninolytic metabolism. Phanerochaete chrysosporium was inoculated onto thin sections of wood. RNA was purified from colonized material after 40 and 96 hours. Single read 100 bp Illumina runs were performed.
Project description:Using Chromatin immunoprecipitation combined with Selective Isolation of Chromatin Associated Proteins (ChIP-SICAP), we define the chromatin-bound proteins co-localized with CTCF in mouse embryonic stem cells (ES) and neuronal stem (NS) cells. We uncover the proteins that are differentially co-localized with CTCF in ES and NS cells. We show the link between the underlying role of RNA-binding proteins with consolidation of chromatin structure during mammalian development.
Project description:The experiment contains ChIP-seq data for Acinetobacter baumannii strain AB5075 encoding 3xFLAG tagged H-NS. Experiments were done with or without ectopic expression of the truncated H-NS-39 protein (corresponding to the H-NS multimerization surface). The strain was grown at 37 degrees in LB medium and crosslinked with 1 % (v/v) formaldehyde. After sonication, to break open cells and fragment DNA, immunoprecipitations were done using anti-FLAG antibodies against. Libraries were prepared using DNA remaining after immunoprecipitation.