Cold tissue dissociation

Kidneys were dissociated using a modified version of the published protocol described in [18]. Based on the weight, in a pre-cooled Miltenyi C-tube, a protease solution (5 mM CaCl₂ [Invitrogen; AM9530G], 10 mg/mL B. Licheniformis protease [Sigma; P5380], 125 U/mL DNase I [Sigma; D5025], 1xDPBS) was prepared for each kidney.

The kidneys were then minced on ice into a smooth paste using a scalpel. The minced kidney was transferred into 4–6 mL of the protease solution (dependent on weight) and triturated using a 1 mL pipette for 15 s every 2 min for a total of 8 min.

Following trituration, the C-tubes were placed onto a Miltenyi gentleMACS octo dissociator in a cool room (4 °C), and the m_brain_03 program was run twice in succession. Once complete, the samples were triturated for 15 s every 2 min on ice for an additional 16 min using a 1-mL pipette. A total of 10 μL of each sample was then loaded into a hemocytometer to assess whether tissue dissociation was complete. Complete tissue dissociation was also checked by examining under the microscope and confirming the absence of visible tissue chunks. The dissociated cells were transferred to a 15-mL centrifuge tube and 3 mL of ice-cold PBS + 10%FBS [Gibco; A3160401] was added.

The cell suspension was centrifuged at 1200g for 5 min at 4 °C. The supernatant was removed and the pellet was resuspended in 2 mL of PBS + 10%FBS. The cells were then filtered through a 70-μm cell strainer, which was subsequently rinsed with 2 mL of PBS + 0.01% BSA. The cells were then centrifuged again at 1200g for 5 min at 4 °C followed by removal of the supernatant and resuspension of the pellet in 5 mL of PBS + 0.01%BSA. The cells were then filtered through a 40-μm cell strainer, which was subsequently rinsed with 2 mL of PBS + 0.01% BSA. The cells were again centrifuged at 1200g for 5 min at 4 °C followed by removal of the supernatant and resuspension of the cells in 5 mL of PBS + 0.04%BSA. The cells were counted and checked for viability using the ReadyProbes Blue/Red Kit on the Countess II FL. The cells were further diluted to a concentration of 700 cells/μL with PBS/0.04%BSA and loaded directly onto a 10x chip (A/B depending on experiment) and isolated using the 10x Chromium controller. The remaining cells were either methanol fixed or cryopreserved. Cell viability is available in Additional file 18.