FACS sorting

BD Aria series or a FACS Melody sorter (BD Biosciences) equipped with a 384-well plate stage was used for single-cell sorting. Two sorting strategies (unbiased and biased) were performed in this study. First, to sort all cell types, we stained viable cells using CellTracker, CMFDA-Green (1:1000, Thermo Fisher), and then unbiasedly sorted dye-positive cells to 384-well plates (Supplementary Fig. ^17a). In general, glomerular cells from an adult mouse were sufficient to sort 1–2 plates and cells from each human kidney biopsy consisting of 10–15 glomeruli were able to sort 250–380 cells. Second, to sort certain cell groups, a biased method was also performed. For instance, to enrich MLCs, CD45⁺ immune cells and CD31⁺ endothelial cells were stained and then were excluded during sorting. Briefly, glomerular cells from six wt mice were pooled and incubated in 50 µl of sorting buffer containing mouse antibodies CD45-PE-CF594 and CD31-APC on ice for 15 min followed by washing. Cells positive for the live cell dye and negative for CD45 and CD31 were gated for sorting (Supplementary Fig. ^17b). By this design, glomerular cells could completely exclude Cd45⁺ immune cells, but we still found Pecam1⁺ endothelial cells after scRNA-seq, probably due to enzymatic destruction of CD31 epitopes. To sort EGFP⁺ cells isolated from two Pdgfrb-EGFP mice, we gated cells positive for DRAQ5 (1:2000, Thermo fisher) and EGFP for sorting. As a control for autofluorescence, kidney cells from wt mice were used (Supplementary Fig. ^17c). Similarly, we sorted podocytes positive for both tdTomato and CMFDA-Green. The flow cytometry analysis software BD FACSDiva v8.0.2 installed in the BD Aria or FACSChorus v1.1 in the BD FACS Melody sorter was used for data analysis as well as single-cell sorting.