NextSeq 500 sequencing; Massively parallel single-cell RNA-sequencing of K14E7 transgenic and wildype C57BL/6 mice
Massively parallel single-cell RNA-sequencing of K14E7 transgenic and wildype C57BL/6 mice
Ear skin from 10-12-week-old mice (n=2 per strain) was split into dorsal and ventral parts and incubated with 2.5 μg/μL Dispase II (Roche, Basel, Switzerland) for 1 h at 37°C. Epidermis and dermis were separated with closed forceps. The epidermis was further homogenized and digested with 1 μg/μL of collagenase D (Roche, Basel, Switzerland) and 0.2 μg/μL of DNase, (Roche, Basel, Switzerland) for 1 h at 37°C. Digested samples were passed through a 0.7 μm filter (BD Falcon, San Jose, USA) to generate a single cell suspension for staining and sorting. Single cell suspensions of digested epidermis samples were incubated with Fc Block (5 μg, rat anti-mouse CD19/CD32, clone 93, eBioscience) diluted in PBS for 30 min on ice. Samples were subsequently incubated with APC-conjugated rat anti-mouse CD45.2 antibodies (0.5 μg, Clone 104, Biolegend) diluted in PBS + 2% serum + 2 mM EDTA for 30 min on ice. Prior to sorting, cells were labelled with 7-AAD (0.25 μg, eBioscience). Live CD45- cells (7-AAD- and APC- or PE-Cy7-) were sorted using the BD ARIA Fusion sorter at 12 psi with a 100 μm nozzle. ~300,000-500,000 events/cells were collected per sample. scRNA-seq was performed in duplicate for wild-type and K14E7 transgenic animals. The 10X Genomics Chromium instrument (10X Genomics) 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 (v1; 10X Genomics; PN-120233) as per the manufacturer's instructions.  Library construction performed as per 10X Genomic Single Cell 3' RNA-seq protocol version 1.