GSM8389326: IRR300 scRNAseq; Mus musculus; RNA-Seq
For single-cell suspension preparation, freshly collected mouse kidneys were rinsed twice with RPMI1640 (Gibco), minced into 3-5 mm3 pieces, and subjected to enzymatic digestion using Multi Tissue Dissociation Kits (130-110-204, Miltenyi Biotec) for 30 min under rotation at 250 rpm/min. Afterward, the cell suspension was filtered and centrifuged at 300 g at 4°C. The cell pellet was collected, resuspended in 1 mL RBC lysis buffer (Sigma) to remove RBCs, and then washed twice with RPMI1640. Cell viability was assessed using Trypan Blue (Gibco), and dead cells were removed using magnetic bead separation with a Dead Cell Removal Kit (130-090-101, Miltenyi Biotec) if the cell viability was less than 85%. At least 20,000 cells per sample were applied to a single-cell master mix with lysis buffer and reverse transcription reagents, following the User Guide of 10X Genomics Chromium Single Cell 3' Reagent Kits (10X Genomics). The libraries were subjected to high-throughput sequencing on a NovaSeq6000 platform.