characteristics.text: 2024-01-01
characteristics.characteristic: ENA first public
characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
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: public
characteristics.characteristic: INSDC status
characteristics.text: ERS20394167
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-14205:LUMC0216_G2G2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: LUMC0216iAPOL
characteristics.characteristic: cell_line
characteristics.text: human iPSC-derived kidney organoid cells
characteristics.characteristic: cell_type
characteristics.text: not collected
characteristics.characteristic: collection date
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: IFNγ treated kidney organoids were harvested and dissociated to single cells as previously reported (Koning M et al. NPJ Regen Med 2022). Briefly, 3 organoids from each cell line were placed in Collagenase I buffer consisting of 600U/mL collagenase Type 1(Worthington) and 0.75 U/mL DNAse (Sigma Aldrich) in HBSS with calcium and magnesium (Thermo Fisher Scientific) and incubated in a water bath at 37 °C for 40min with pipetting up and down 2-3 times with 1mL pipet. The cell suspensions were centrifuged at 300g for 7min and the supernatant was removed. Cells were resuspended in TryPLE buffer consisting of 5U/mL DNAse I (Sigma Aldrich) and 4 ug/mL heparin (Sigma Aldrich) in 80% TrypLE select 10x (Thermo Fisher Scientific) in DPBS (Thermo Fisher Scientific) and incubated at 37 °C for 5 min with repeated pipetting. The dissociation was stopped by adding cold HBSS with 10% FCS. The single cell suspension was centrifuged at 400g and the cell pellet was resuspended in PBS with 0.1% BSA. Single dose of 25ng/mL IFN-γ was added to kidney organoids on d7+17 and the medium was refreshed without IFN-γ on d7+19. The organoids were harvested on d7+20. Nucleic acid extraction was performed in single cells using the Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 and Chromium Next GEM Chip G Single Cell Kit according to manufacturer’s instructions. The single cell suspensions obtained from dissociated kidney organoids were converted to barcoded scRNA-seq libraries using the Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 and Chromium Next GEM Chip G Single Cell Kit. 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 equivalent
characteristics.characteristic: developmental stage
characteristics.text: APOL1 nephropathy
characteristics.characteristic: disease
characteristics.text: APOL1 G2G2 genotype
characteristics.characteristic: genotype
characteristics.text: not collected
characteristics.characteristic: geographic location (country and/or sea)
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: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: LUMC0216_G2G2
characteristics.characteristic: title