GSM5706854: Urine AKI 2; Homo sapiens; RNA-Seq
All steps were performed on ice. Urine was collected as either a voided specimen or from a urinary catheter. Samples were immediately transferred to a biosafety level (BSL) 2+ laboratory for processing. Urine samples were transferred to a 50mL conical tube and centrifuged at 1000x g for 10 minutes at 4 degrees celsius. Cell pellets were washed with ice-cold PBS, filtered through a 40µm filter, and centrifuged again. Live cells were purified using the MACS Debris Removal Kit (Miltenyi Biotec) followed by the EasySep Annexin V Dead Cell Removal Kit (StemCell). Briefly, cells were resuspended in ice-cold PBS and mixed with debris removal solution. Cold PBS was overlaid on the mixture and centrifuged at 300x g for 10 minutes at 4 degrees celsius. The top two phases were aspirated and then remaining cells were washed with PBS. Cells were resuspended and mixed with Dead Cell Removal Cocktail, Biotin Selection Cocktail, then RapidSpheres before separation in an EasySep magnet. Cells were washed and resuspended in 52µL of PBS (no calcium or magnesium) + .04% BSA (Fisher Scientific) for scRNAseq processing. Purified cells were immediately processed using the Chromium 3' Single Cell RNA sequencing kit (10X Genomics) according to the manufacturer's instructions.