For each sample, a 4-mm punch biopsy was obtained following local anesthesia and was placed immediately into 10 mL of RPMI on ice. Initially, skin biopsies were incubated in 5mL of a 0.4% Dispase II solution (Roche Inc.) at 37°C for 1 hour with vigorous shaking. The dermis and epidermis were then carefully separated using forceps and transferred to separate tubes for additional processing. Epidermal samples were placed in 3mL of 0.25% Trypsin and 10U/mL DNAse for 30 minutes at 37°C. Trypsin was neutralized with 3mL of fetal calf serum (FCS), and the tissue was passed through a 70-micron nylon cell strainer which was washed with 5mL of RPMI. Epidermal cells were then pelleted at 300xg for 10 minutes and counted. Dermal samples were minced with a scalpel and incubated in a solution of 0.4% collagenase type II and 10 U/mL DNAse for 2 hours at 37°C with agitation. The cell suspension was passed through a 70-micron cell strainer and washed with 5mL of RPMI. Cells were pelleted at 300xg for 10 minutes, resuspended in 1mL of RPMI and counted. MACS enrichment for CD1a+ cells was performed for epidermis from three RR patients. Once a single-cell suspension was obtained, we utilized Seq-Well, a massively-parallel scRNA-Seq platform for clinical samples, to capture the transcriptome of single cells. Full methods on implementation of Seq-Well are available in Gierahn et al. or on the Shalek Lab website (www.shaleklab.com). Briefly, 10-15,000 cells were loaded onto a functionalized-polydimethylsiloxane (PDMS) array preloaded with uniquely-barcoded mRNA capture beads (Chemgenes; MACOSKO-2011-10). After cells had settled into wells, the array was then sealed with a hydroxylated polycarbonate membrane with pore sizes of 10 nm, facilitating buffer exchange while confining biological molecules within each well. Following membrane-sealing, subsequent buffer exchange permits cell lysis, mRNA transcript hybridization to beads, and bead removal before proceeding with reverse transcription. The obtained bead-bound cDNA product then underwent Exonuclease I treatment (New England Biolabs; M0293M) to remove excess primer before proceeding with second strand synthesis. Single cell RNA sequencing
GEO Accession
GSM4581231