ERC000011
ENA-CHECKLIST
2022-08-05T00:28:55Z
ENA-FIRST-PUBLIC
2022-08-05T00:28:55Z
ENA-LAST-UPDATE
SAMEA12997830
External Id
VIB-KU Leuven Center for Cancer Biology
INSDC center name
2022-08-05T00:28:55Z
INSDC first public
2022-08-05T00:28:55Z
INSDC last update
public
INSDC status
ERS10596726
SRA accession
E-MTAB-11429:KOR2
Submitter Id
ArrayExpress
broker name
hiPSC reporter MAFB:mTagBFP2
cell_line
kidney organoid
cell_type
human
common name
Protocols: Kidney organoids were generated from human induced pluripotent stem cells using previously published protocols (van den Berg et al., Stem Cell Reports 2018). On d7+12 of differentiation, organoids were either transplanted inside the coelomic cavity of day 4 chicken embryos (White Leghorn eggs (sp: Gallus gallus domesticus) have been used), or maintained in vitro. One day (day 7+13) and 8 days (d7+20) after transplantation, transplanted organoids and respective untransplanted (in vitro) controls were dissociated to single cells using 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) followed by 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). After dissociation, cells were resuspended in 1mL PBS + 0,1% BSA. All cells from organoids from the same condition were pooled (d7+13 untransplanted: 94 organoids, transplanted: 75 bisected organoids (transplanted in 150 chicken embryos); d7+20 untransplanted: 95 organoids, transplanted: 47 bisected organoids (transplanted in 93 chicken embryos)). 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.
description
neonate
developmental stage
normal
disease
in vivo transplanted into coelomic cavity of Gallus gallus domesticus embryos
growth condition
pool of 150 organoids
individual
Homo sapiens
organism
Homo sapiens
organism
prepuce of penis
organism part
induced pluripotent stem cell
progenitor cell type
Homo sapiens
scientific_name
male
sex
KOR2
title