GSM8170104: Kidney Explants D9, Replicate 1, scRNA-Seq; Mus musculus; RNA-Seq
Kidney explants were dissociated using 10 mg/mL Bacillus licheniformis cold active protease (Sigma P5380) mixed with 2.5 mg/mL collagenase type 2 (Worthington, #LS00417) and 125 U/mL DNase I (Worthington, #LS002058) in DPBS at 12°C for 30 min (1 mL total reaction volume). The digestion mix was mixed twenty times with P-200 pipette tips/5 min. The dissociation reaction was terminated by mixing with 1mL of 20% fetal bovine serum (FBS) in DPBS to a final concentration of 10%FBS. The cells were filtered through a pre-wetted 40-um strainer (Falcon), and 1 mL of DPBS was used to wash cells off the strainer. The 3 mL dissociated cell mix was combined with 3 mL of AutoMACS Running Buffer (Miltenyl Biotec, 130-091-221) and cells were briefly pelleted at 300g for 5 mins at 4°C. The cell pellet was then resuspended in AutoMACS Running Buffer, with 14 μM DAPI and 5 μM DRAQ5 added freshly. Singlet live cells (DAPI- and DRAQ5+) were selected by fluorescence-activated cell sorting (FACS) on a BD SORP FACSAria IIu (ARIA #1) cell sorter. Single cells were pelleted to concentrate at 300G x 5 mins. The targeted number of cells then proceeded to drop-seq separation on the 10X machine (Chip B). Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10 X Genomics).