Single cell RNA-seq on kidney allograft urine samples
Single cell RNA-seq on kidney allograft urine samples
Freshly voided urine was passed through a 40 µm cell strainer and centrifuged at 400 g for 10 minutes at room temperature. The cell pellet was resuspended in 2% BSA-DPBS and subjected to Ficoll-Paque density gradient centrifugation at 1312 g for 20 minutes at room temperature. Cells were then collected and washed with 2% BSA-DPBS, followed by centrifugation at 400 g for 10 minutes at room temperature. The pellet was resuspended in 0.5% BSA-DPBS, filtered through a 30 µm cell strainer, and centrifuged again at 200 g for 6 minutes at room temperature. Finally, the cells were resuspended in 0.5% BSA-DPBS. We used the 10x Genomics' Chromium Single Cell 3' Reagent Kit v3 (first three samples) or v3.1 (subsequent nine samples), according to the manufacturer's instructions, for the library preparation.