DNBSEQ-G400 sequencing; scRNAseq of human iPSC-derived kidney organoids transplanted in the coelomic cavity of chicken embryos compared to untransplanted controls
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.