A key limitation in single cell genomics is generating a high-quality single cell suspension that contains rare or difficult to dissociate cell types and is free of RNA degradation or transcriptional stress responses. Samples with unpredictable availability or that must be collected at several timepoints present additional challenges. Using adult mouse kidney, we compared single-cell RNA sequencing (scRNA-seq) data generated using DropSeq with snRNA-seq data generated from nuclei using sNuc-DropSeq, DroNc-seq and 10X Chromium. We validated snRNA-seq on fibrotic kidney from day 14 unilateral ureteral obstruction (UUO). Overall design: Dropseq, sNucDropseq, DroNcSeq and 10X Chromium were used to profile mouse healthy and fibrotic kidneys
Advantages of Single-Nucleus over Single-Cell RNA Sequencing of Adult Kidney: Rare Cell Types and Novel Cell States Revealed in Fibrosis.
Subject
View SamplesFibrosis is the common final pathway of virtually all chronic injury to the kidney. While it is well accepted that myofibroblasts are the scar-producing cells in the kidney, their cellular origin is still hotly debated. The relative contribution of proximal tubular epithelium and circulating cells including mesenchymal stem cells, macrophages and fibrocytes to the myofibroblast pool remains highly controversial. Using inducible genetic fate tracing of proximal tubular epithelium we confirm that proximal tubule does not contribute to the myofibroblast pool. However, in parabiosis models in which one parabiont is genetically labeled and the other is unlabeled and undergoes kidney fibrosis, we demonstrate that a small fraction of genetically labeled renal myofibroblasts derive from the circulation. Single cell RNA-Sequencing confirms this finding but indicates that these cells are circulating monocytes, express few extracellular matrix or other myofibroblast genes and do express many proinflammatory cytokines. We conclude that this small circulating myofibroblast progenitor population contributes to renal fibrosis by paracrine rather than direct mechanisms. Overall design: Single cell RNA-seq was performed on FACS-sorted PDGFRB+CD45- and PDGFRB+CD45+ cell populations
Parabiosis and single-cell RNA sequencing reveal a limited contribution of monocytes to myofibroblasts in kidney fibrosis.
Age, Subject
View SamplesAfter-ripening induced seed dormancy release in wheat is associated with mRNA oxidation.
Integrated analysis of seed proteome and mRNA oxidation reveals distinct post-transcriptional features regulating dormancy in wheat (Triticum aestivum L.).
Specimen part
View SamplesGlioma-associated oncogene homolog-1 (Gli1)-positive resident mesenchymal stem cell-like cells are the predominant source of kidney myofibroblasts in fibrosis, but investigating Gli1-positive myofibroblast progenitor activation is hampered by the difficulty of isolating and propagating primary cultures of these cells. Using a genetic strategy with positive and negative selection, we isolated Kidney-Gli1 (KGli1) cells that maintain expression of appropriate mesenchymal stem cell-like cell markers, respond to hedgehog pathway activation, and display robust myofibroblast differentiation upon treatment with transforming growth factor-ß (TGF-ß). Coculture of KGli1 cells with endothelium stabilizes capillary formation. Single-cell RNA sequencing (scRNA-seq) analysis during differentiation identified autocrine ligand-receptor pair upregulation and a strong focal adhesion pathway signal. This led us to test the serum response factor inhibitor CCG-203971 that potently inhibited TGF-ß-induced pericyte-to-myofibroblast transition. scRNA-seq also identified the unexpected upregulation of nerve growth factor (NGF), which we confirmed in two mouse kidney fibrosis models. The Ngf receptor Ntrk1 is expressed in tubular epithelium in vivo, suggesting a novel interstitial-to-tubule paracrine signaling axis. Thus, KGli1 cells accurately model myofibroblast activation in vitro, and the development of this cell line provides a new tool to study resident mesenchymal stem cell-like progenitors in health and disease. Overall design: DropSeq on primary culture kidney Gli1+ cells harvasted from 0, 6, 12, and 24 hrs after TGF-beta treatment
A conditionally immortalized Gli1-positive kidney mesenchymal cell line models myofibroblast transition.
Sex, Treatment, Subject
View SamplesComparison of gene expression from subjects who resolved or formed pustules to H.ducreyi.
Dysregulated immune profiles for skin and dendritic cells are associated with increased host susceptibility to Haemophilus ducreyi infection in human volunteers.
No sample metadata fields
View SamplesChlorine is a widely used industrial chemical that is also considered a chemical threat agent. Inhalation of chlorine gas can cause acute injury to the respiratory tract, including the death of airway epithelial cells. Failure to efficiently repair the epithelial damage is associated with long-term respiratory abnormalities, including airway fibrosis. We previously developed a model of airway injury in which mice exposed to chlorine gas exhibit epithelial damage and develop fibrosis in large airways.
Inhibition of chlorine-induced airway fibrosis by budesonide.
Sex, Age, Specimen part, Treatment
View SamplesAcute kidney injury (AKI) is associated with an abrupt loss of kidney function that results in significant morbidity and mortality. Considerable effort has focused around the identification of diagnostic biomarkers and the analysis of molecular events. Most studies have adopted organ-wide approaches that do not fully capture the interplay among different cell types in the pathophysiology of AKI. To extend our understanding of molecular and cellular events in AKI, we developed a mouse line that enables the identification of translational profiles in specific cell types by CRE recombinase-dependent activation of an eGFP-tagged L10a ribosomal protein subunit, and consequently, translating ribosome affinity purification (TRAP) of mRNA populations. By utilizing cell-type specific CRE-driver lines, in this study we identify distinct cellular responses in an ischemia reperfusion injury (IRI) model of AKI. Cell-specific translational expression profiles were uncovered 24 hours after IRI from four populations enriched for distinct anatomical and cellular subgroups: nephron, interstitial cell populations, vascular endothelium, and macrophages/monocytes by Affymetrix microarray.
Cell-specific translational profiling in acute kidney injury.
Sex, Age, Specimen part, Treatment
View SamplesThe long term goal is to define the transcriptional changes that accompany pericyte-to-myofibroblast transition in fibrotic kidney disease. Medullary pericytes are identified by their expression of a eGFPL10a fusion protein whose expression is driven by a Col1a1 promoter. Pericyte-specific RNA is generated by eGFP-affinity purification of polysomes from medullary lysates and then subject to microarray analysis.
Translational profiles of medullary myofibroblasts during kidney fibrosis.
Sex, Specimen part, Time
View Samplesgene expression study on brain and lung under Dip2a regulation to better understand the role of Dip2a gene during mice brain and lung development.
Large genomic fragment deletions and insertions in mouse using CRISPR/Cas9.
Sex, Specimen part, Cell line
View SamplesThis experiment is to examine the effect of PARP inhibitor and Myc shRNA knockdown on transcriptome profiles in MYC-amplified human GBM stem cells MGG4. Overall design: There are totally 4 samples. GBM cell MGG4 expressing scramble shRNA or shRNA targeting Myc were grown in doxycycline (Dox, 1 mg/ml) for 6 days, treated with olaparib (Ola, 10 microM) or DMSO for 24h, and harvested for RNA extraction, followed by RNA sequencing
Myc targeted CDK18 promotes ATR and homologous recombination to mediate PARP inhibitor resistance in glioblastoma.
Cell line, Treatment, Subject
View Samples