NextSeq 500 sequencing; scRNA-seq of CD45+ cells taken from the ear epidermis from K14E7 transgenic and wildype C57/BL6 mice
scRNA-seq of CD45+ cells taken from the ear epidermis from K14E7 transgenic and wildype C57/BL6 mice
Single-cell suspensions of digested epidermis samples were incubated with Fc Block (5 mg, rat anti-mouse CD19/CD32, clone 93; eBioscience, San Diego, CA) diluted in phosphate buffered saline for 30 minutes on ice. Samples were subsequently incubated with APC-conjugated rat anti-mouse CD45.2 antibodies (0.5 mg, clone 104; BioLegend, San Diego, CA) diluted in phosphate buffered saline plus 2% serum plus 2 mmol/L EDTA for 30 minutes on ice. Before sorting, cells were labelled with 7-aminoactinomycin D (AAD) (0.25 mg, eBioscience). Live CD45+ cells (7-AAD+ and antigen-presenting cell+ or PE-Cy7+) were sorted into 100% serum containing 2 mmol/L EDTA using the BD ARIA Fusion sorter (Franklin Lakes, NJ) at 12 p.s.i. with a 100-mm nozzle. Samples were kept chilled throughout the experiment prior to loading onto the 10x Chromium Instrument. Approximately 300,000 events/cells were collected per sample. Flow cytometry analysis of epidermis and dermis is prepared similarly. LCs were analyzed with the following antibodies: PE-Cy7-anti-CD11c (HL3) and PE-anti-CD103 (2E7; BD), PerCP-Cy5.5-anti-CD45 (30-F11), Alexa Fluor® 700-anti-CD11b (M1/70) and APC-anti-EpCAM (G8.8) (BioLegend). Ear skin from 10- to 12-week-old mice (n = 2 per strain) was split into dorsal and ventral parts and incubated with 2.5 mg/ml Dispase II (Roche) for 1 hour at 37oC. Epidermis and dermis were separated with closed forceps. The epidermis was further homogenized and digested with 1 mg/ml of collagenase D (Roche) and 0.2 mg/ml of DNase (Roche) for 1 hour at 37oC. Digested samples were passed through a 0.7-mm ﬁlter (BD Falcon, Franklin Lakes, NJ) to generate a single-cell suspension for staining and sorting. scRNA-seq was performed in duplicate for wildtype, K14E7 and K14E7xRbΔL transgenic animals. The 10x Genomics Chromium instrument was used to partition viable CD45+ cells with barcoded beads, and cDNA from each cell was prepared using the Single Cell 3' Library, Gel Bead and Multiplex Kit (version 2, PN-120233; 10X Genomics) per the manufacturer's instructions. Cell numbers in each reaction were optimized to capture approximately 3,000 cells.  cDNA shearing was performed with a Covaris S2 instrument (Covaris, Woburn, MA) set to produce a target size of 200 bp (intensity = 5, duty cycle = 10%, cycles = 200, time = 120 seconds). Libraries were constructed as per the manufacturer's instructions.