characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2022-06-10T00:18:52Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2022-06-10T00:18:52Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA12941032
characteristics.characteristic: External Id
characteristics.text: UOE
characteristics.characteristic: INSDC center name
characteristics.text: 2022-06-10T00:18:52Z
characteristics.characteristic: INSDC first public
characteristics.text: 2022-06-10T00:18:52Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS10539828
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-11079_2:ZF1
characteristics.characteristic: Submitter Id
characteristics.text: 6
characteristics.unit: month
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: renin lineage cell
characteristics.characteristic: cell type
characteristics.text: zebrafish
characteristics.characteristic: common name
characteristics.text: Protocols: Mesonephric kidneys were isolated into ice-cold Leibowitz medium. Cells were dissociated in medium supplemented with cold activated protease (20mg/ml), DNAse (400units/ml), Liberase (0.085mg/ml), collagenase (2mg/ml) and CaCl2 (5mM), at 6oC for 20 mins with frequent triturition. Samples were transferred to 28.5oC for an additional 10 mins to complete digestion. Digested kidneys were passed through a 40micron sieve prior to centrifugation. Cells were resuspended in PBS with 2% FCS for FAC sorting. A WIK mesonephric kidney was used to control for autofluorescence, and DAPI was used for the live/dead cell count. Samples were sorted for red and green fluorescence, in order to sort renin-expressing cells and smooth muscle cells respectively. Zebrafish were terminally anaesthetised (tricaine methanesulphonate; MS-222) and decapitated. Lower abdomen was removed to expose kidney which was carefully teased away from the spine. Zebrafish were maintained in a Home Office approved establishment at 28.5oC under standard feeding and light regimen. Renin gene was targetted using CRISPR Cas9 technology. Briefly, an sgRNA, specifically targeting exon 2 of the renin gene, was complexed with tracr-RNA and Cas9 protein and injected into a one-cell embryo. Surviving fish were genotyped by tail clip and targeted fish were crossed onto WIKs. The genotype of F1 progeny was determined by sequencing across the target site and fish heterozygous for an 8bp deletion in renin exon 2 were crossed to generate ren-/- knockout fish. Nucleic acid was extracted using the Chromium Single Cell 3' Library and Gel Bead kit v2 (10X Genomics, PN-120237) and the Chromium Single Cell A Chip kit (10X Genomics, PN-120236) according to manufacturers instructions. Nucleic acid library was constructed using the Chromium Single Cell 3' Library and Gel Bead kit v2 (10X Genomics, PN-120237) and the Chromium Single Cell A Chip kit (10X Genomics, PN-120236) according to manufacturers instructions.
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: ren:RFP; acta2:EGFP
characteristics.characteristic: genotype
characteristics.text: ~9-12 kidneys pooled
characteristics.characteristic: individual
characteristics.text: juxtaglomerular cell
characteristics.characteristic: inferred cell type
characteristics.text: Danio rerio
characteristics.characteristic: organism
characteristics.text: Danio rerio
characteristics.characteristic: organism
characteristics.text: kidney
characteristics.characteristic: organism part
characteristics.text: wild type
characteristics.characteristic: phenotype
characteristics.text: Danio rerio
characteristics.characteristic: scientific_name
characteristics.text: mixed
characteristics.characteristic: sex
characteristics.text: WIK
characteristics.characteristic: strain
characteristics.text: ZF1
characteristics.characteristic: title