title: GSM9038760: all mix sample 5; Mus musculus; RNA-Seq
library_construction_protocol: Euthanized mice were perfused with chilled 1× PBS via the left ventricle. After removing the renal capsule and fat, kidneys were minced into approximately 1 mm3 cubes and digested using Multi-Tissue Dissociation kit (Miltenyi, 130-110-201). Up to 250 mg of 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. Digestion was deactivated by 10% fetal bovine serum (FBS). The suspension was passed through a 70 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 and washed again. Cells were resuspended in PBS and passed through a 40 μm cell strainer. Cells were resuspended in staining buffer (1× PBS, 1% BSA, 2 mM EDTA) for further analysis. Kidneys were collected from C57BL/6 mice subjected to ischemia-reperfusion injury (IRI) or left untreated as controls (n=3 per condition). Single-cell suspensions were prepared from pooled kidneys (3 mice per condition), and live leukocytes were enriched by fluorescence-activated cell sorting (FACS) using SYTOX™ Blue exclusion and forward/side scatter gating. Cell viability was confirmed by Trypan Blue exclusion (>80%). Live cells were fixed using the Evercode™ Cell Fixation Kit (V2) (Parse Biosciences) and stored at –80 °C. Fixed cells were thawed and processed with the Evercode™ WT (V2) kit for single-cell library preparation according to manufacturer protocols. Libraries were sequenced at ~50,000 reads per cell on a NovaSeq 6000 platform (Illumina) with 5% PhiX spike-in, performed by Novogene.
identifiers.namespace: GEO
identifiers.id: GSM9038760_r1