Search results for "Single-Cell Analysis"
showing 10 items of 34 documents
Assessment of computational methods for the analysis of single-cell ATAC-seq data
2019
Abstract Background Recent innovations in single-cell Assay for Transposase Accessible Chromatin using sequencing (scATAC-seq) enable profiling of the epigenetic landscape of thousands of individual cells. scATAC-seq data analysis presents unique methodological challenges. scATAC-seq experiments sample DNA, which, due to low copy numbers (diploid in humans), lead to inherent data sparsity (1–10% of peaks detected per cell) compared to transcriptomic (scRNA-seq) data (10–45% of expressed genes detected per cell). Such challenges in data generation emphasize the need for informative features to assess cell heterogeneity at the chromatin level. Results We present a benchmarking framework that …
Configurable low-cost plotter device for fabrication of multi-color sub-cellular scale microarrays.
2014
We report on the construction and operation of a low-cost plotter for fabrication of microarrays for multiplexed single-cell analyses. The printing head consists of polymeric pyramidal pens mounted on a rotation stage installed on an aluminium frame. This construction enables printing of microarrays onto glass substrates mounted on a tilt stage, controlled by a Lab-View operated user interface. The plotter can be assembled by typical academic workshops from components of less than 15 000 Euro. The functionality of the instrument is demonstrated by printing DNA microarrays on the area of 0.5 squared centimeters using up to three different oligonucleotides. Typical feature sizes are 5 μm diam…
From non-excitable single-cell to multicellular bioelectrical states supported by ion channels and gap junction proteins: Electrical potentials as di…
2019
Endogenous bioelectric patterns within tissues are an important driver of morphogenesis and a tractable component of a number of disease states. Developing system-level understanding of the dynamics by which non-neural bioelectric circuits regulate complex downstream cascades is a key step towards both, an evolutionary understanding of ion channel genes, and novel strategies in regenerative medicine. An important capability gap is deriving rational modulation strategies targeting individual cells' bioelectric states to achieve global (tissue- or organ-level) outcomes. Here, we develop an ion channel-based model that describes multicellular states on the basis of spatio-temporal patterns of …
Deformation-induced transitional myofibroblasts contribute to compensatory lung growth
2016
In many mammals, including humans, removal of one lung (pneumonectomy) results in the compensatory growth of the remaining lung. Compensatory growth involves not only an increase in lung size, but also an increase in the number of alveoli in the peripheral lung; however, the process of compensatory neoalveolarization remains poorly understood. Here, we show that the expression of α-smooth muscle actin (SMA)—a cytoplasmic protein characteristic of myofibroblasts—is induced in the pleura following pneumonectomy. SMA induction appears to be dependent on pleural deformation (stretch) as induction is prevented by plombage or phrenic nerve transection (P < 0.001). Within 3 days of pneumonectomy, …
Heterogeneity of circulating CD8 T-cells specific to islet, neo-antigen and virus in patients with type 1 diabetes mellitus
2018
Auto-reactive CD8 T-cells play an important role in the destruction of pancreatic β-cells resulting in type 1 diabetes (T1D). However, the phenotype of these auto-reactive cytolytic CD8 T-cells has not yet been extensively described. We used high-dimensional mass cytometry to phenotype autoantigen- (pre-proinsulin), neoantigen- (insulin-DRIP) and virus- (cytomegalovirus) reactive CD8 T-cells in peripheral blood mononuclear cells (PBMCs) of T1D patients. A panel of 33 monoclonal antibodies was designed to further characterise these cells at the single-cell level. HLA-A2 class I tetramers were used for the detection of antigen-specific CD8 T-cells. Using a novel Hierarchical Stochastic Neighb…
Single cell detection of latent cytomegalovirus reactivation in host tissue
2011
The molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need to be identified, ideally at the earliest possible time point in the process. To this end, mouse cytomegalovirus (MCMV) reporter mutants were designed to simultaneously express the red fluorescent protein mCherry and the secreted Gaussia luciferase (Gluc). Whereas Gluc can serve to assess infection at the level of individual mice by measuring luminescence in blood samples or by in vivo imaging, mCherry fluorescence offers the advatage of detection of infection at the single cell level. To visualiz…
Single-cell analysis of the ventricular-subventricular zone reveals signatures of dorsal and ventral adult neurogenesis
2021
The ventricular-subventricular zone (V-SVZ), on the walls of the lateral ventricles, harbors the largest neurogenic niche in the adult mouse brain. Previous work has shown that neural stem/progenitor cells (NSPCs) in different locations within the V-SVZ produce different subtypes of new neurons for the olfactory bulb. The molecular signatures that underlie this regional heterogeneity remain largely unknown. Here, we present a single-cell RNA-sequencing dataset of the adult mouse V-SVZ revealing two populations of NSPCs that reside in largely non-overlapping domains in either the dorsal or ventral V-SVZ. These regional differences in gene expression were further validated using a single-nucl…
Co-option of Neutrophil Fates by Tissue Environments
2020
Classically considered short-lived and purely defensive leukocytes, neutrophils are unique in their fast and moldable response to stimulation. This plastic behavior may underlie variable and even antagonistic functions during inflammation or cancer, yet the full spectrum of neutrophil properties as they enter healthy tissues remains unexplored. Using a new model to track neutrophil fates, we found short but variable lifetimes across multiple tissues. Through analysis of the receptor, transcriptional, and chromatin accessibility landscapes, we identify varying neutrophil states and assign non-canonical functions, including vascular repair and hematopoietic homeostasis. Accordingly, depletion…
Cell population analysis of the adult murine subependymal neurogenic lineage by flow cytometry
2021
Summary This protocol provides a flow-cytometry-based procedure to classify and isolate all cells of the adult rodent subependymal zone (SEZ) neurogenic lineage, without the need for reporter mice, into different cell populations, including three neural stem cell (NSC) fractions with molecular signatures that are coherent with single-cell transcriptomics. Additionally, their cycling behavior can be assessed by means of 5-ethynyl-2′-deoxyuridine (EdU) incorporation. Our method allows the isolation of different NSC fractions and the functional assay of their cycling heterogeneity and quiescence-activation transitions. For complete details on the use, execution, and outcomes of this protocol, …
Laser Microdissection of the Alveolar Duct Enables Single-Cell Genomic Analysis
2014
Complex tissues such as the lung are composed of structural hierarchies such as alveoli, alveolar ducts, and lobules. Some structural units, such as the alveolar duct, appear to participate in tissue repair as well as the development of bronchioalveolar carcinoma. Here, we demonstrate an approach to conduct laser microdissection of the lung alveolar duct for single-cell PCR analysis. Our approach involved three steps. The initial preparation used mechanical sectioning of the lung tissue with sufficient thickness to encompass the structure of interest. In the case of the alveolar duct, the precision-cut lung slices were 200µm thick; the slices were processed using near-physiologic conditions…