title: scRNAseq for characterization of Kitl-expressing stromal cells in ear skin
design: scRNAseq for characterization of Kitl-expressing stromal cells in ear skin
library_construction_protocol: After sacrifice, ears from two KitlGFP mice (18 weeks old littermate males from the same cage) were isolated. Ears from each mouse were separated into dorsal and ventral sides and both sides were finely minced in 500 µl OptiMEM (ThermoFisher) medium.  For digestion, 500 µl of digestion mix containing LiberaseTM DL (0.5 mg/ml), Collagenase IV (2.4 mg/ml), DNAse I (2 mg/ml) was added and samples were incubated for 45 min at 37°C, 1200 rpm shaking. During incubation, samples were resuspended every 15 min by pipetting. Digestion was stopped by pouring samples together through a 70 µm cell strainer and pooling them into 5 ml ice-cold OptiMEM medium with 10 % FCS and 1 % penicillin (10 U/ml)/streptomycin (10 µg/ml). After centrifugation, pellets were resuspended in 1 ml erythrocyte lysis buffer (155 mM NH4Cl2, 10 mM KHCO3, 0.1 mM EDTA in H2O) and incubated for 10 min at RT. Lysis buffer was washed off with FACS buffer (PBS + 2 % FCS V/V).  For subsequent sorting, the pellet was first incubated for 10 min at 4°C with anti-mouse CD16/32 (Fc block, Becton Dickinson; 25 µg/ml in FACS buffer) followed by 20 min incubation at 4°C with DAPI (0.5 µg/ml) and anti-CD31 AlexaFluor 647 (25 µg/ml) in FACS buffer. After incubation, cells were washed with FACS buffer and resuspended in PBS with 2 % BSA and 2 mM EDTA for sorting. Sorting was performed using a FACS Aria III (Becton Dickinson). DAPI-negative CD31-negative GFP-positive cells were sorted into DNA low binding Eppendorf tubes containing ice-cold PBS with 2 % BSA. Sorted cells in PBS supplemented with 2% BSA were pelleted at 300g for 5 min at 4 °C. Cells were resuspended in PBS with 0.5% BSA to a maximum cell concentration of 395 cells/µl and placed on ice. Cell viability was assessed by trypan blue staining. A cell suspension aliquot (2 µl) was diluted 1:2 in 0.4% trypan blue solution, incubated for 2 minutes on a microscope slide and analyzed under a microscope. All samples for scRNA-seq analysis showed more than 90% viable cells.  Single cell RNA-sequencing was performed on the 10X Genomics platform using the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 dual index (10x Genomics, PN-1000268) following the manufacturer's instructions. A total of 43 µl of cell suspension was loaded onto a chip G according to the manufacturer's instructions, aiming for a targeted cell recovery of up to 10,000 cells. PCR cycles for cDNA and final library generation were adjusted according to the target cell recoveries. The quality of the obtained cDNA and final libraries was assessed by capillary electrophoresis (Fragment analyzer, HS NGS Fragment Kit, Agilent).