Warm tissue dissociation

Kidneys were dissociated using the Multi-tissue dissociation kit 2 from Miltenyi Biotec [130-110-203] as per manufacturers’ instruction, with minor variations. Once the weight of the kidney was determined, the kidney was quartered and placed into a gentleMACS C-tube [Miltenyi Biotech; 130-096-334] containing the enzyme mix described in the kit’s protocol. The tube was centrifuged briefly, then placed onto the gentleMACS octo dissociator (Miltenyi Biotech), and the 37C_Multi_E program was run after attaching the heating elements. Following completion of the program, the tube was briefly centrifuged. Complete tissue dissociation was checked by examining under the microscope and confirming the absence of visible tissue chunks.

The homogenate was filtered through a 70-μm cell strainer [Greiner; 54,207] into a 50-mL centrifuge tube [Greiner; 227,270], the strainer was then rinsed with 15 mL of PBS. The cell suspension was centrifuged at 400g for 10 min; once complete, the supernatant was removed and the pellet was resuspended in 5 mL of PBS + 0.04% BSA [Sigma; A7638]. The cell suspension was then filtered through a 40-μm strainer [Greiner; 542,040], which was subsequently rinsed with 2 mL of PBS + 0.04% BSA. The cells were again centrifuged at 400g for 10 min. The supernatant was then removed, and the pellet was resuspended in 5 mL of PBS + 0.04% BSA. Cell count and viability was estimated using the Countess II FL (Thermo Fisher) and the ReadyProbes Blue/Red kit [Invitrogen; {"type":"entrez-nucleotide","attrs":{"text":"R37610","term_id":"795066","term_text":"R37610"}}R37610]. The cells were then diluted to 700 cells/μL and were immediately loaded onto a 10x chip A and processed on the 10x Chromium controller. The remaining cells were then either methanol fixed or cryopreserved. Cell viability is available in Additional file 18.