GSM5987780: pooled sample, patients P008; P009; P010; P012, CD45+ urine cells_HTO; Homo sapiens; RNA-Seq
Samples were collected either as first morning void urine or via urinary catheter (using the pooled urine output of 4 hours) and stored on ice. Incubation with Actinomycin D for 30 min on ice was applied to prevent subsequent alteration of transcriptomes followed by blocking Fc receptors using human FcR blocking reagent (Miltenyi, Bergisch Gladbach, Germany). Cells were then incubated with fluorescent dyes and fluorochrome-conjugated monoclonal antibodies for fluorescence-activated cell sorting (FACS). The following antibodies and dyes were used for cell labeling: Calcein AM (BD Biosciences, San Jose, CA, USA, 564061), Hoechst 33342 (BD Biosciences, San Jose, CA, USA, 561908), AnnexinV-PE (Biolegend, San Diego, CA, USA, 640908), CD45-APC/Vio770 and CD66b-PE/Vio770 (both Miltenyi, 130-110-635 and 130-119-768). Cells were washed, filtered through a 70 Âµm cell strainer and labeled with To-Pro3 iodide (T3605, Life technologies, Eugene, Oregon) for dead cell discrimination immediately prior to sorting.  Samples were sorted using a Sony MA900 (Sony, Tokyo, Japan) with a 100Âµm nozzle into phosphate buffered saline (PBS) / 1 % BSA gating To-Pro3-AnnexinV- Hoechst33342+CalceinAM+ singlets for obtaining single viable urinary cells. Urinary granulocytes, which make up a large fraction of the urinary cellular signal (median 58% in FACS gating) but for which a renal tissue origin is not verifiable, and which may interfere with downstream analysis, were excluded. Relative single cell counts in sorted suspensions were counted using a MACSQuant Analyzer (Miltenyi). Cells were centrifuged, resuspended, and re-counted on a hemocytometer. Cell suspensions were subjected to single-cell sequencing following the 10x Genomics protocol for Chromium Next GEM Single Cell 3â€™ v3.1 chemistry targeting between 1000 - 10000 cells depending on sample cell count. According to manufacturer recommendation of  Chromium Next GEM Single Cell 3Ê¹ GEM, Library & Gel Bead Kit v3.1