GSM7868162: FGF23_1h, scRNAseq; Mus musculus; RNA-Seq
Murine kidneys were transported in RPMI1640 (Corning), on ice immediately after surgical procurement. Kidneys were rinsed with PBS and minced into eight sections. Each sample was then enzymatically and mechanically digested for 30 min (Multi-Tissue Dissociation Kit two and GentleMACS dissociator/tube rotator, Miltenyi Biotec). The samples were prepared per protocol 'Dissociation of mouse kidney using the Multi Tissue Dissociation Kit 2' with the following modifications: After termination of the program 'Multi_E_2', we added 10 mL RPMI1640 (Corning) and 10% BSA (Sigma-Aldrich) to the mixture, filtered and homogenate was centrifuged (300 g for 5 min at 4°C). The cell pellet was resuspended in 1 mL of RBC lysis buffer (Sigma), incubated on ice for 3 min, and cell pellet washed three times (300 g for 5 min at 4°C). Annexin V dead cell removal (Stem Cell Technologies) was performed using magnetic bead separation after final wash, and the pellet resuspended in RPMI1640/BSA 0.04%. Viability and counts were assessed using Trypan blue (Gibco) and brought to a final concentration of 1 million cells/mL, exceeding 80% viability as specified by 10x Genomics processing platform. 10x Genomics protocol for chromium single cell 3' reagent kits V3