GSM7337040
Harvested P0 mouse kidneys were dissected into smaller pieces in cold Dulbecco's phosphate-buffered saline (DPBS Ca2+-/Mg2+-, Gibco 14190-250). For cell isolations, the Bacillus licheniformis cold active protease (CAP) protocol was used to dissociate kidney tissue into cells as previously described in (Ransick et al., 2019). Briefly, tissue was lysed in the DPBS lysis buffer, with the final concentration of a 2.5 mg/ml type 2 collagenase (Worthington, #LS00417), 7.5 mg/ml B. licheniformis protease (Creative Enzymes, NATE-0633), and 125 U/ml DNase I (Worthington, #LS002058) at 12°C. After 30 mins of digestion, the reaction was terminated with 20% fetal bovine serum (FBS) in DPBS. Cells were then strained through coarse 40 um cell strainers (Falcon, #352340) and pelleted at 364g in a swinging bucket centrifuge at 6oC. Cells were resuspended in cold AutoMACs Running Buffer (AMB, Miltenyi Biotec) and strained again with fine mesh 40um strainer (VWR, #76327-098). Live cells with DNA staining (DAPI-/DRAQ5+) were sorted by ARIA II FACS (BD Biosciences) and pelleted at 350g for 10mins then resuspended in ice cold AMB. Chromium_SingleCell_3'_ReagentKits_v2/v3_UserGuide (CG00052, CG000183)