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One human embryo of w16 was isolated and the kidney dissected in cold saline solution (0.9% NaCl, Versylene Fresenius, FranceThe obtained kidney was decapsulated and kept on ice in dissociation buffer (DPBS + Penicillin 100U/ml + Streptomycin 0.1 mg/ml) before cutting it into 1 – 2 mm pieces. The pieces were washed 3 times with washing solution (Advanced DMEM F12 supplemented with: ITS commercial solution (Insulin – Transferrin – Selenium), Glutamax, Penicillin 100 U/ml and Streptomycin 0.1 mg/ml) with brief centrifugation (160g) in order to remove as many red blood cells as possible. The washed kidney tissue was then incubated with digestion solution (Trypsin/EDTA solution 0.25% and Collagenase-II 280 U/ml) and incubated overnight at 4°C. The next day, the digestion solution was removed, the kidney was rinsed with washing solution and incubated with washing solution for 30 min at 37°C with agitation. Subsequently, the sample was sequentially passed through sterile cell-strainers of 100, 70 and 40 µm pore size with the help of washing solution. The cells were then centrifuged, counted and viability was measured to be 78% (trypan blue assay) before proceeding with single-cell RNA sequencing library preparation.10X library: Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 3' Reagent Kit, Version 2 Chemistry (10x Genomics) according to the manufacturer's protocol. Libraries were sequenced on a NextSeq500 in Mid Output mode using a version 2, 150 cycles kit (Illumina).; Human fetal embryos were isolated and the kidney dissected in cold saline solution (0.9% NaCl, Versylene Fresenius, France). The obtained kidneys were decapsulated and kept on ice in dissociation buffer (DPBS + Penicillin 100U/ml + Streptomycin 0.1 mg/ml) before cutting it into 1 – 2 mm pieces. The pieces were washed 3 times with washing solution (Advanced DMEM F12 supplemented with: ITS commercial solution (Insulin – Transferrin – Selenium), Glutamax, Penicillin 100 U/ml and Streptomycin 0.1 mg/ml) with brief centrifugation (160g) in order to remove as many red blood cells as possible. The washed kidney tissues were then incubated with digestion solution (Trypsin/EDTA solution 0.25% and Collagenase-II 280 U/ml) and incubated overnight at 4°C. The next day, the digestion solution was removed, the kidneys were rinsed with washing solution and incubated with washing solution for 30 min at 37°C with agitation. Subsequently, the samples were sequentially passed through sterile cell-strainers of 100, 70 and 40 µm pore size with the help of washing solution. The cells were then centrifuged, counted and viability was measured to be 78% (trypan blue assay) before proceeding with single-cell RNA sequencing library preparation.10X library: Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 3' Reagent Kit, Version 2 Chemistry (10x Genomics) according to the manufacturer's protocol. Libraries were sequenced on a NextSeq500 in Mid Output mode using a version 2, 150 cycles kit (Illumina). |
