GSM8194407: d23_kidney_organoids; Homo sapiens; RNA-Seq
Kidney organoids were incubated for 30 min at 6oC in cold active protease (DPBS(+), 5 mg/mL Bacillus Licheniformis Protease, 125 U/mL DNase) . Each cell suspension was diluted with fluorescence-activated cell sorting (FACS) buffer (DPBS(-), 0.5% FBS, 2 mM EDTA) + 125 U/mL DNase, centrifuged at 300 x g for 10 min at 4oC after which the supernatant was removed. This step was repeated twice after which the suspension was passed through a 35 μm filtered tube (Falcon) to prepare a cell suspension containing single cells. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Cells from kidney organoids were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.