characteristics.text: 2024-01-01
characteristics.characteristic: ENA first public
characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: SAMEA114785899
characteristics.characteristic: External Id
characteristics.text: Department of Internal Medicine (Nephrology) & Einthoven Laboratory of Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands
characteristics.characteristic: INSDC center name
characteristics.text: 2023-12-03T10:23:55Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS17207276
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-13595:KOR10
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: hiPSC reporter MAFB:mTagBFP2
characteristics.characteristic: cell_line
characteristics.text: kidney cell
characteristics.characteristic: cell_type
characteristics.text: not collected
characteristics.characteristic: collection date
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: Untransplanted and transplanted organoids generated using the hiPSC-MAFB line were dissociated to single cells at d7+13 and d7+20 of differentiation. At d7+13, 94 untransplanted and 75 bisected transplanted organoids (150 chicken embryos) were dissociated, and at d7+20, 49 untransplanted and 24 bisected transplanted organoids (48 chicken embryos) were used. Organoids were incubated in a collagenase I buffer consisting of 600U ml-1 collagenase Type I (Worthington) and 0,75U ml-1 DNAse (Sigma Aldrich) in HBSS with calcium and magnesium (Thermo Fisher Scientific) for 40 minutes in a water bath at 37°C with repeated pipetting. Cell suspensions were centrifuged for 7 minutes at 300G and resuspended in a TrypLE buffer consisting of 5U ml-1 DNAse I (Sigma Aldrich) and 4µg ml-1 heparin (Sigma Aldrich) in 80% TrypLE select 10x (Thermo Fisher Scientific) in DPBS (Thermo Fisher Scientific). They were incubated for another 5 minutes at 37°C before dissociation was stopped through addition of cold HBSS+/+ with 10% FCS and further diluted with HBSS+/+. Cells were centrifuged for 5 minutes at 400G and resuspended in wash buffer (PBS + 0,1% BSA). After dissociation, all cells from organoids from the same condition were pooled and diluted in wash buffer. Viable ECs were identified and sorted on a FACSAria II or III cell sorter using a staining for human CD31 combined with a viability dye: rganoid cells were incubated with CD31-PE (1:100, BD Biosciences 555446) and viability dye eFluor 780 (1:5000, ThermoFisher Scientific 65-0865-14) in FACS buffer (PBS + 0,1% BSA + 2mM EDTA) for 30 minutes at 4⁰C. Cooked cells (20 minutes at 60⁰C) and cells from dissociated organoids derived from the LUMC0072iCTRL01 line were used for compensation. Viable endothelial cells (d7+13 untransplanted: 23060, transplanted: 6843. D7+20 untransplanted: 6697, transplanted: 36444) were sorted using a FACS Aria II 3L or FACS Aria III 4L (BD Biosciences) and collected in wash buffer. Cells were centrifuged for 6 minutes at 400G and resuspended in 10X buffer (PBS + 0,04% BSA). Cell number and viability were assessed using a Bio-Rad TC20 cell counter after staining with Trypan Blue. Due to the low number of sorted cells from transplanted d7+13 and untransplanted d7+20 organoids, these were not counted. The single cell suspensions were converted to barcoded scRNA-seq libraries with a targeted cell recovery of 6000 cells / condition. Nucleic acid extraction was performed in single cells using the Chromium Single Cell 3’ v3 Library, Gel Bead & Multiplex Kit and Chip Kit (10x Genomics) according to manufacturer’s instructions. The single cell suspensions obtained from dissociated kidney organoids were converted to barcoded scRNA-seq libraries using the Chromium Single Cell 3’ Library, Gel Bead & Multiplex Kit and Chip Kit (10x Genomics). Samples were processed with 10x Genomics’ V3 barcoding chemistry. Cells from each sample were treated with the same master mix and in the same reaction vessel, by processing every single sample in a single well of a PCR plate.
characteristics.characteristic: description
characteristics.text: fetal stage
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: not collected
characteristics.characteristic: geographic location (country and/or sea)
characteristics.text: in vivo transplanted
characteristics.characteristic: growth condition
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: kidney organoid
characteristics.characteristic: organism part
characteristics.text: induced pluripotent stem cell
characteristics.characteristic: progenitor cell type
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: KOR10
characteristics.characteristic: title