GSM5462525: Mouse kidney scRNA-seq_IRI_short_3d_sample1; Mus musculus; RNA-Seq
Euthanized mice were perfused with chilled 1x PBS via the left ventricle. Kidneys were harvested, minced into approximately 1 mm3 cubes and digested using Multi Tissue dissociation kit (Miltenyi, 130-110-201). The tissue was homogenized using 21G and 26.5G syringes. Up to 0.25 g of the tissue was digested with 50 µl of Enzyme D, 25 µl of Enzyme R and 6.75 µl of Enzyme A in 1 ml of RPMI and incubated for 30 min at 37 °C. Reaction was deactivated by 10% FBS. The solution was then passed through a 40 µm cell strainer. After centrifugation at 400 g for 5 min, cell pellet was incubated with 1 ml of RBC lysis buffer on ice for 3 min. Cell number and viability were analyzed using Countess AutoCounter (Invitrogen, C10227). This method generated single-cell suspensions with >80% viability. Libraries were generated using Chromium Single Cell 3' Reagent Kits v3 (10X Genomics, PN-1000092) and Chromium i7 Multiplex Kit (10X Genomics, PN-120262) according to the manufacturer's manual. Quality control for constructed library was performed by Agilent Bioanalyzer High Sensitivity DNA kit (Agilent Technologies, 5067-4626) for qualitative analysis. Quantification analysis was performed by Illumina Library Quantification Kit (KAPA Biosystems, KK4824).