GSM8997885: Pkd2 mutant-1; Mus musculus; RNA-Seq
scRNA-seq analysis was performed on Pkd2 and DKO mutant kidneys at 16 and 26 weeks post-tamoxifen induction, respectively. Kidneys (n=3 per mutant group, n=1 per each control group, all females; 8 total mice) were isolated and processed using a modified cold-activated protease digestion. Birefly, kidneys were minced and digested in 1 ml of 2.5 mg/ml type II collagenase (Worthington, catalog no. LS004176), 7.5 mg/ml B. licheniformis cold-activated protease (Creative Enzymes, catalog no. NATE-0633), and 125 U/ml DNase in D-PBS for approximately 45 minutes at 12°C. After digestion, kidney tissues was individually passed through 70-µm cell-strainers (Falcon; BD Biosciences), yielding single-cell suspensions. Cells were centrifuged at 1300 rpm for 5 minutes at 4°C, resuspended in ACK red blood cell lysis buffer, and incubated at 37°C for 5 minutes. After 5 minutes, 10 ml of RPMI was added to each tube, cells were spun at 1300 rpm for 5 minutes at 4°C, and cells were resuspended in 1% BSA with FC blocking solution (1:200; BioXcell, catalog no. BE0307) for 30 minutes on ice. After blocking, cells were spun and incubated with CD45 (catalog nos. 12–0451, 30-F11; eBioscience) and Fixable Aqua Dead Cell Stain for 30 mins. After staining, cells were spun, washed with 1% BSA, and  ready for sorting. About 30000 CD45+  cells from each samples were sorted via FACSAriaII sorter. After dilited to disired concentration, the single-cell suspensions were loaded onto the Chromium Controller (10x Genomics) to generate single-cell Gel Bead-in-Emulsions (GEMs) using the GEM-X Universal 3' Gene Expression v4 4-plex On-chip Multiplexing (OCM) kit, following manufacturer's protocols. Sequencing libraries were constructed from amplified cDNA and sequenced on an Illumina platform at a depth of 20,000 reads per cell. Data processing, including demultiplexing, alignment, and gene expression quantification was conducted (Cell Ranger pipeline, 10x Genomics, v3.1). Sequencing libraries were constructed from amplified cDNA and sequenced on an Illumina platform at a depth of 20,000 reads per cell. Data processing, including demultiplexing, alignment, and gene expression quantification was conducted (Cell Ranger pipeline, 10x Genomics, v3.1).