GSM6656497
Dissociation of kidney samples into single cell suspensions was performed using dissected cortex from necropsy and CUK samples or else from approximately 50% of a biopsy sample. Tissue pieces were minced into 1-2mm3 and digested by type IV collagenase (Sigma, C5138-500mg; 1mg/mL in 10mL of HBSS [ThermoFisher, MT-20023CV]). The resulting single cell suspensions were passed through a 100 m cell strainer (Miltenyi Biotec, 130-098-463), centrifuged and then passed through a 70 m cell strainer (Miltenyi Biotec, 130-098-462) and centrifuged again. RBC lysis was performed by resuspending these pellets with 4 mL RBC lysis buffer (Roche, 11814389001) and placed on ice for 2 minutes. RBC lysis reaction was quenched with 25mL 20% fetal bovine serum in HBSS and subsequently centrifuged again, pellets resuspended in HBSS and refiltered through 70 m filters. A dead cell removal step was performed using Dead Cell Removal Kit following the manufacturer's instructions (Miltenyi Biotec, 130-090-101). After dead cell removal, cells were counted and resuspended at ~600-1200cells/ L. These suspensions were applied to the 10X Genomics Chromium Next GEM Single Cell 3' v3.1 kit to prepare scRNA-seq libraries, per manufacturer's instructions (10X Genomics, 1000121).