title: GSM9640483: L2HGDH_OE_Kidney_4 (scRNA-seq); Mus musculus; RNA-Seq
library_construction_protocol: After mice (P11) were euthanized, they were perfused via the left ventricle with ice-cold Hanks' balanced salt solution (HBSS) to remove blood, and the kidneys were harvested and minced with a sterile razor blade into small pieces (< 1 mm diameter). The tissue was digested in HBSS containing Liberase TM (0.30 mg/mL) (Roche, catalog no. 291963), Hyaluronidase (10 μg/mL) (Sigma, catalog no. H4272), and DNase1 (20 μg/mL) for 40 minutes at 37 °C on a shaker (300 rpm, Eppendorf Thermomixer 5355). The suspension was centrifuged at 300 × g for 5 min at 4 °C, and the supernatant was discarded. The cells were washed twice with 2 mL cold PBS (ThermoFisher, catalog no. 14190250) and pelleted. The pellets were resuspended in 5 mL of 0.25% Trypsin with DNase1 (20 μg/mL) and incubated for 15 min at 37 °C on a shaker for further digestion. Trypsin was inactivated with 2 mL 10% FBS in PBS after digestion, followed by centrifugation at 300 × g for 5 min at 4 °C. Cell pellets were washed once again with 10% FBS, followed by resuspending the cell pellets in ice-cold PBS supplemented with 0.01% BSA. The cell suspension was filtered sequentially through 70 μm and 40 μm strainers, pelleted, and resuspended in 0.01% BSA. Cell number and viability were analyzed using the Nexcelom Cellometer Auto2000 with the acridine orange (AO) and propidium iodide (PI) fluorescent staining method. All resuspensions used wide-bore tips. Libraries were then generated using the 10X Genomics Universal 3' V4 kit, according to the manufacturer's instructions, using a 10X Genomics Chromium X Controller.