Gene expression profile at single cell level of vessel associated kidney NKp46+ ILC from healthy and nephritic NZB/W lupus prone mice
Gene expression profile at single cell level of vessel associated kidney NKp46+ ILC from healthy and nephritic NZB/W lupus prone mice
In total, we analyzed NKp46+ ILC, isolated from 10 healthy young, and 6 nephritic mice, by scRNA-seq. Kidneys were harvested, minced and placed into RPMI 1640 Medium with GlutaMAX (Gibco) with collagenase IV (Worthington Biochemical, 570 U/ml) and DNase I (Roche, 12,5 U/ml). Tissue digestion was performed using the gentleMACS™ Dissociator and incubation with the enzymes at 37°C for 20 minutes. Single cell suspensions were obtained by passing cells through a 70-µm and 40-µm strainer strainer and washing once with FACS buffer. Following centrifugation at 500g for 5 minutes at 4°C, erythrocytes were removed by centrifugation in Percoll gradient. For sorting of NKp46+NK1.1+ ILC leukocytes were further enriched by Percoll gradient centrifugation (Cytiva). The pellet was resuspended in 40% Percoll layered over 80% Percoll prior to density centrifugation (1306 g 20 min at 20ºC with no brake). Cells were plated in 96-well V-bottom plates. FcγRIII/II were blocked with purified anti-CD16/32 (FcγRIII/II, eBioscience) diluted 1:250 for 15 minutes at 4o degrees. Antibody mixes were added and incubated at 4°C in the dark for 30 minutes, then incubated with streptavidin conjugates for 20 minutes. For surface stains DAPI was added to stain dead cells before analysis.  Cell sorting was performed using a BD Biosciences FACSAria II cell sorter achieving a purity rate of > 98%. For kidney NK1.1+ NKp46+ ILC, 5' gene expression libraries were generated using Chromium Next GEM Single Cell 5' Kit v2 (10X Genomics) according to the manufacturer's instruction. Quality control of cDNA and final libraries was done using Bioanalyzer High Sensitivity DNA Analysis (Agilent) and the KAPA library quantification kit. Libraries were sequenced using NextSeq 2000 (Illumina).