GSM9553656: Pat0_rep1_urine_GEX; Homo sapiens; RNA-Seq
Paired samples of heparinized venous blood and freshly voided non-morning urine (usually 2nd urine of the day; processed less than one-hour post excretion) were collected from patients who were hospitalized at the Charité - Universitätsmedizin Berlin due to acute lupus flares with renal involvement. After dipstick analysis to exclude urinary tract infection urine was diluted 1:2 in Annexin binding buffer (ABB) with 1 % BSA and 2 µg/ml Actinomycin D (ActD). Peripheral blood was treated with 2 µg/ml ActD and both body fluids were incubated for 30 min on ice. ActD should preserve transcriptomes from alteration ex vivo. Urine pellets from centrifugation (300 g, 7 min, 4 °C) and PBMC isolated by density gradient centrifugation (600 g, 20 min, 25 °C) with Ficoll-Paque were subjected to red blood cell lysis with ammonium chloride buffer when necessary. Subsequent to 20 min incubation on ice with AnnexinV-FITC (Biolegend, 640906 or Miltenyi, 130-093-060), FCγR blocking reagent and fluorochrome conjugated mAb against human CD3 (APC-eFluor780, SK7, eBioscience, 47-0036-42), CD4 (PE-Vio770, REA623, Miltenyi, 130-113-227) and CD8 (PE or APC, GN11/134D7, DRFZ in-house) cell pellets were filtered through 30 µm mesh, washed at least 10X volume of ABB, resuspended in 2 ng/ml DAPI -containing ABB and sorted by FACS on machines from BD (FACSAria or FACSAriaII) with 70 µm nozzles for DAPI- AnnexinV- CD3+ and CD4+ or CD8+ events. Cell sorters were maintained by the FCCF at DRFZ in Berlin. Events from urine were sorted into one tube. CD4+ or CD8+ T cell events from blood were sorted separately for and mixed subsequently at the CD4-to-CD8 ratio equivalent to that from paired urine samples. ScRNA-seq was conducted subsequently when absolute sorted event count from urine was 3000 or above. Library construction was conducted according to protocols Chromium Single Cell 3' Library and Gel Bead Kit v2 (PN-120267) for Pat0 and Pat1, Chromium Single Cell 5' Library and Gel Bead Kit v1 (PN-1000014) for Pat2 (replicate one) and Pat3 5, Chromium Next GEM Single Cell 5' Library and Gel Bead Kit v1.1 (PN-1000167) for Pat2 (replicate two) and Pat6. Libraries of Pat2 6 were split and separately processed for sequencing of gene expression (GEX) and immune receptors V(D)J, here T cell receptor (TCR). Intermediate and final library quantity and quality was assessed with Qubit and Bioanalyzer2100 (Agilent).