GSM8968338: scRNA-seq, blood; Mus musculus; RNA-Seq
Kidneys were dissected into small fragments and incubated at 37 °C for 40 min in RPMI 1640 Medium (Thermo Fisher) containing 10% heat inactivated FBS, 1% P/S, 0.4 µg/ml Collagenase D (Roche, Basel, Switzerland) and 0.2 µg/ml DNAse (Roche). Following this, tissue was dissociated using the GentleMACS Dissociator and C-Tubes (both Miltenyi Biotec) and leukocytes were isolated using a gradient centrifugation with 37 % Percoll (GE Healthcare). After the centrifugation, cells were washed with DPBS (Gibco). T cells were sorted by Fluorescence-activated cell sorting (FACS) for CD3+ T cells. From the spleen and blood of nephritic mice, erythocytes were removed by a lysis buffer containing 155 mM NH4Cl, 10 mM KHCO3, and 10 µM EDTA at a pH of 7.2. After washing, CD45+ cells were sorted by FACS for CD3+ and CD3- cells. Twenty thousands of CD45+CD3+ and CD45+CD3- cells were used for scRNA-seq analysis. Cells were loaded on a 10x Chromium single cell instrument. All steps were performed according to standard protocol of the Chromium single cell 5' v2 kit to generate high-quality cDNA libraries.