title: GSM6805689: Pod-I_Ppo-1, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: The digestion buffer was a mixture of Dulbecco PBS with collagenase type II (3 mg/ml, Worthington), ProNase E (1.5 mg/ml, Millipore), DNAse (62.5 U/mL, Takara), and 5 mM calcium chloride. Kidneys were harvested, minced into 1 mm3 cubes, and digested using digestion buffer with shaking at 37 °C for 20 minutes. Then, the digestion mixture was incubated in a thermomixer for 5 minutes at 37 °C with shaking at 1200 RPM. The solution was successively passed through 70- and 30-μm cell strainers. The solution was then centrifuged at 300 ×g for 5 minutes, and the resulting cell pellet was diluted with DMEM– high-glucose media (Gibco) with 10% FBS (Sigma‒Aldrich). The solution was passed through 30-μm cell strainers three times, and a single-cell suspension was obtained. The single-cell suspension was loaded onto a chip on the 10× Chromium Single Cell instrument (10× Genomics). Bar coding and cDNA amplified were performed using Chromium Single Cell 3' Reagent Kits v3.1 (10× Genomics) according to the manufacturer's instructions. Sequencing is performed on an MGI tech DNBSEQ-G400 at Azenta Japan Corp. Cell Ranger v3.1 (10× Genomics) software with mouse mm10 reference sequence in prebuild Cell Ranger reference packages was used for initial data processing. After initial data processing, all processing steps were performed using Seurat v3.