Project description:3 sets of CFZ-exposed bacteria were compared to 3 sets of bacteria treated with vehicle (DMSO) for 24 hours in an aerated drug killing model.
Project description:In this study, we performed a systematic analysis of ortho nitrobenzyl alcohol (oNBA) chemistry for integration into novel reagents for chemical crosslinking-mass spectrometry.
Project description:Biomarker identification for diagnosis of systemic juvenile idiopathic arthritis (SJIA), an auto-inflammatory disease that presents with prolonged fevers. Disease vs. healthy control
Project description:In order to identify unexpected, or indeed previously uncharacterized genes may be important in sex- or gonad development, we developed a custom cDNA microarray represent 3837 unique transcripts of Scylla paramamosain derived from our EST project. Thirty-nine putative transcripts were observed to differentially expressed in testis and ovaries (P<0.05). Two-condition experiment, Ovary vs. Testis. Biological replicates: 3 Ovaries, 3 Testis, 2 dye-swaps.
Project description:Existing protocols for full-length single-cell RNA sequencing (scRNA-seq) produce libraries of high complexity (thousands of distinct genes) with outstanding sensitivity and specificity of transcript quantification. These full-length libraries have the advantage of allowing probing of transcript isoforms, are informative regarding single nucleotide polymorphisms, and allow assembly of the VDJ region of the T- and B-cell receptor sequences. Since full length protocols are mostly plate-based at present, they are also suited to profiling cell types where cell numbers are limiting, such as rare cell types during development for instance. A disadvantage of these methods has been the scalability and cost of the experiments, which has limited their popularity as compared to droplet-based and nanowell approaches. Here, we describe an automated protocol for full-length scRNA-seq, including both an in-house automated SMART-seq2 protocol, and a commercial kit-based workflow. We discuss these two protocols in terms of ease-of-use, equipment requirements, running time, cost per sample and sequencing quality. By benchmarking the lysis buffers, reverse transcription enzymes and their combinations, we propose an optimized in-house automated protocol with dramatically reduced cost. These pipelines have been employed successfully for several research projects allied with the Human Cell Atlas initiative (www.humancellatlas.org) and are available on protocols.io.
Project description:YbjN, an enterobacteria-specific protein, is a multicopy suppressor of ts9 temperature sensitivity in Escherichia coli. Microarray study revealed that the expression level of ybjN was inversely correlated with the expression of flagellar, fimbrial and acid resistance genes. Over-expression of ybjN significantly down-regulated genes involved in the citric acid cycle, glycolysis, the glyoxylate shunt, oxidative phosphorylation, and amino acid and nucleotide metabolism. On the other hand, over-expression of ybjN up-regulated toxin-antitoxin modules, the SOS responsive pathway, cold shock proteins and starvation-induced transporter genes. Our results collectively suggest that YbjN may play important roles in regulating bacterial multicellular behaviors, metabolism and survival under various stress conditions in Es. coli. A total of 8 samples were analyzed: E. coli wild type strain (2 replicates); E. coli ybjN mutant strain (3 replicates); E. coli ybjN over-expression strain (3 replicates).
Project description:The p53 transcription factor family consists of the three members p53, p63 and p73. Both p63 and p73 exist in different isoforms that are well characterized. Isoforms have also been identified for p53 and it has been proposed that they are responsible for increased cancer metastasis. In contrast to the p63 and p73 isoforms, which do not contain truncations in folded domains, most of the p53 isoforms contain only parts of either the DNA binding domain or the oligomerization domain. To better understand the effect of p53 isoforms in cancer we provide here a comprehensive biochemical characterization. With the exception of the Δ40p53α isoform none of the other variants can bind to DNA with high affinity and none can upregulate transcription. Probing with antibodies, DARPins and other interaction partners confirmed that isoforms harboring deletions in the DNA binding domain cannot interact specifically with them, but instead are bound to chaperones and other factors known to interact with misfolded proteins. Expression of isoforms with deletions in the DNA binding domain results in upregulation of cellular chaperones. If the expression level surpasses a threshold, the chaperone system can no longer keep these isoforms soluble resulting in aggregation and co-aggregation with other factors.
Project description:The BldC gene is required for the formation of aerial hyphae in Streptomyces venezuelae. It is a 68 amino acid DNA-binding protein related to the MerR family of transcription factors. BldC deletion strains are bald because they initiate sporulation prematurely, omitting the formation of aerial hyphae altogether. This RNA-Seq experiment was carried out to determine and quantify effect of BldC on the expression of all genes in the S. venezuelae genome after 10 and 14 hours of culture.
Project description:The basic experiment is a comparison of gene transcript levels of Streptomyces coelicolor M145 and its wblA deletion mutant at 6 timepoints (24, 36, 48, 60, 72 and 84 hours) encompassing vegetative growth, aerial growth, and sporulation on solid medium. The wblA mutant is morphologically defective, most of its aerial hyphae failing to show any sign of sporulation-directed attributes, and also overproduces some antibiotics. The microarray analysis was aimed to reveal the major transcriptional changes underpinning or associated with this pleiotropic phenotypic change. Using a fairly stringent cut-off, 291 genes were found to be affected, including developmental genes, antibiotic biosynthetic genes, and genes for primary metabolism. Some genes were over-expressed and others under-expressed in the mutant. Although the largest effects were mostly at timepoints corresponding to aerial growth and early sporulation, some genes were most strongly influenced at earlier timepoints. M145 vs wblA deletion mutant. 6 timepoints (24, 36, 48, 60, 72 and 84 hours) encompassing vegetative growth, aerial growth, and sporulation, were analysed. Each timepoint comprised 2 technical replicates each of 3 biological replicates. RNA was isolated from triplicated cultures grown on cellophane overlays on MM + mannitol after 24, 36, 48, 60, 72 and 84 hours of incubation. Samples of the RNA were converted to Cy3-labelled cDNA in vitro, and mixed with Cy5-labelled genomic DNA. Each mixture was hybridised to two glass slide microarrays each carrying oligonucleotide probes corresponding to 99% of the genes in the annotated S. coelicolor genome. Thus RNA from each timepoint was compared to the same batch of genomic DNA and the comparison used to arrive at expression values of genes. These expression values were then used to arrive at differential expression between M145 and the wblA deletion mutant for all genes at all six timepoints.
Project description:Whole genome mapping of protein-DNA interactions can be performed by coupling chromatin immunoprecipitation (ChIP) with high-throughput sequencing (ChIP-Seq). However, current methods require large amounts of starting materials which precludes their application to rare cell types. Here, we combine a high-sensitivity ChIP assay with a novel library preparation procedure to map histone modifications in as few as 10,000 cells. We apply the technique to acquire genome-wide chromatin maps for an enriched population of hematopoietic progenitors, and thereby gain insight into their developmental program. An optimized ChIP protocol for small number of cells and a novel Librray preperation methods for high throughput sequencing of picograms amount of ChIP DNA.