GSM8346287: Patient 06, Kidney; Homo sapiens; RNA-Seq
Single cell suspensions were obtained from human kidney biopsies by enzymatic digestion in RPMI 1640 medium with collagenase D at 0.4 mg/ml (Roche, 11088858001) and deoxyribonuclease I (DNase I; 10 μg/ml; Sigma-Aldrich, 10104159001) at 37°C for 30 min followed by dissociation with gentle MACS (Miltenyi Biotec). Leukocytes from blood samples were separated using Leucosep tubes (Greiner Bio-One, 10349081). Cells were stained with fluorochrome-conjugated antibodies from BioLegend and BD Biosciences, CD45 BV510 (HI30), CD3 BV785 (OKT3), CD4 BV650 (RPA-T4), CD8 APC-R700 (RPA-T8), CXCR3 Pe/Dazzle (G025H7), CCR6 PerCP-Cy5-5 (G034E3). Cells were also stained with a dead cell stain (Molecular Probes, L10119) to exclude dead cells from analysis. FACS was performed on a FACSAria Fusion cell sorter (BD Biosciences). ScRNA-seq of human samples from the kidney and peripheral blood was performed from FACS-sorted CD3 positive T cells using Single Cell 3' v2 (10x Genomics) according to manufacturer's instructions. Libraries were sequenced aiming at 50,000 reads per cell on an Illumina HiSeq4000. libaries were constructed according to manufacturer specifications Libraries were sequenced aiming at 50,000 reads per cell on an Illumina HiSeq4000.