GSM9137966: K3_7d; Rattus norvegicus; RNA-Seq
kidneys were perfused via the abdominal aorta with ice-cold phosphate-buffered saline (PBS) for blood clearance. The three kidneys per group were pooled, minced into 1–2 mm³ fragments, and digested in collagenase (17018029/17104019, eBioscience™) at 37°C for 45 min. The digestate was filtered through a 40-μm cell strainer. Erythrocytes were lysed using 1 ml ice-cold RBC Lysis Buffer (C3702, Beyotime) for 5 min, followed by PBS washing to obtain a single-cell suspension. The prepared single-cell suspension was counted and examined by microscopy. Cells were incubated with anti-CD45-APC antibody (17-0461-82, eBioscience™) at 4°C for 30 min. During the last 5 min of incubation, DAPI (1:1000 dilution, 62248, eBioscience™) was added and mixed. After incubation, cells were washed once with RPMI-1640 medium (12633020, eBioscience™) via centrifugation (300 × g, 5 min), resuspended, and filtered through a 40-μm cell strainer. The final suspension was transferred to flow cytometry tubes and kept protected from light on ice. Pre-prepared collection tubes containing RPMI-1640 with 10% FBS were loaded into the sorting chamber for CD45⁺ cell sorting. After sorting, the fluidics system was flushed with sterile ultrapure water. A subset of sorted cells was re-analyzed for quality control, and flow cytometry data were recorded. The target cell suspension was transferred from flow tubes to 15-mL centrifuge tubes, pelleted by centrifugation, counted, examined microscopically, and processed for single-cell library preparation. The cell suspension was loaded into Chromium microfluidic chips with 3' (v2 or v3, depends on project) chemistry and barcoded with a 10× Chromium Controller (10X Genomics). RNA from the barcoded cells was subsequently reverse-transcribed and sequencing libraries constructed with reagents from a Chromium Single Cell 3' v2(v2 or v3, depends on project) reagent kit (10X Genomics) according to the manufacturer's instructions. Sequencing was performed with Illumina according to the manufacturer's instructions (Illumina).