Project description:We performed single-cell transcriptomics analyses (10x Chromium 3' v3.1) using dissociated total kidney cells to identify the difference in gene expression patterns and signaling pathways between male and female mouse kidneys after selective induction of ferroptotic stress in renal tubular epithelial cells.
Project description:Unbiased single-cell RNA-sequencing in freshly-dissociated cells from healthy and stenotic mouse kidneys identified stenotic-kidneys epithelial cells undergoing both mesenchymal transition and senescence.
Project description:We performed single nucleus ATAC (snATAC-seq) to generate cell-type-specific chromatin accessibility atlas of the mouse kidneys with ischemia-reperfusion injury. The single nucleus RNA-seq of the same samples were previously published (GEO139107)
Project description:Kidneys from multiple litters of Six2GFP+ E14.5 mouse embryos were pooled into three replicate tubes and dissociated in parallel. Six2GFP+ cells were isolated and processed for single cell sequencing using 10x Genomics technology. This resulted in a dataset of 7844 single cells representing the nephron progenitor population of the mouse kidney.
Project description:We performed Drop-seq using dissociated total kidney cells to identify cell types, gene expression patterns and signaling pathways in mouse kidneys after IRI.
Project description:Ferroptosis is a form of regulated cell death driven by the iron-dependent accumulation of oxidized polyunsaturated-fatty-acid-containing phospholipids (PUFA-PLs). Three key molecular features of ferroptosis are peroxidation of PUFA-PLs, accumulation of redox-active ferrous iron, and defective lipid peroxide repair (Dixon and Stockwell, 2019). However, there is currently no reliable way to selectively stain ferroptotic cells in tissue sections to characterize the extent of ferroptosis in animal models of disease and in patient tissue samples. We sought to address this gap by immunizing mice with membranes from lymphoma cells treated with the ferroptosis inducer piperazine erastin (PE), and screening ~4,750 of the resulting monoclonal antibodies generated for their ability to selectively detect cells undergoing ferroptosis. We found that one antibody, termed 3F3 Ferroptotic Membrane Antibody (3F3-FMA), was effective as a selective ferroptosis-staining reagent using immunofluorescence (IF). The antigen of 3F3-FMA was identified by immunoprecipitation and mass spectrometry as the human transferrin receptor 1 protein (TfR1), which imports iron from the extracellular environment into cells. We validated this finding with several additional anti-TfR1 antibodies, and compared them to other potential ferroptosis-detecting reagents via immunofluorescence staining, using fluorescence microscopy and flow cytometry. We found that anti-TfR1 and anti-malondialdehyde (MDA) adduct antibodies were effective at reliably staining ferroptotic tumor cells in numerous cell culture and tissue contexts. In summary, these findings suggest that anti-TfR1 antibodies can be used to selectively label cells undergoing ferroptosis.
Project description:To elucidate changes in cellular populations in the ischemic acute kidney injury, we performed single cell RNAseq on mouse kidneys following ischemic reperfusion injury and a sham surgery. Mice were subjected to bilateral clamping of the renal pedicule for 30 minutes. After 6-7 hours of reperfusion the kidneys were removed and processed by a MACs cell isolation protocol and dead cell removal (PMID:33448928) , sorted on a 10X Chromium platform version 3 and libraries generated. Sequencing was performed on an Illumina NovaSeq 6000.