Homo sapiens
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.
disease
normal
organism
Homo sapiens
organism
Homo sapiens
collection date
not collected
scientific_name
Homo sapiens
common name
human
organism part
kidney organoid
developmental stage
fetal stage equivalent
genotype
wild type G0G0 genotype (corrected G2G2 line)
isolate
not applicable
cell_line
LUMC0216iAPOL
cell_type
human iPSC-derived kidney organoid cells
geographic location (country and/or sea)
not collected