GSM9629405: Epi-EED; Homo sapiens; OTHER
FACS isolation of epidermal cells from human skin was performed as follows: Skin samples were collected, and the adipose layer was scraped off using a surgical scalpel and washed with PBS. Samples were incubated in the enzymes, followed by the Epidermis Dissociation Kit manufacturer's protocol (Miltenyi Biotec). The cell pellet was washed twice with DPBS (Thermo Fisher Scientific) before staining with cell-surface markers. The cells were stained with 1:200 PE-CD324(E-cadherin, BD Bioscience) and 1:200 FITC-α6-integrin (Thermo Fisher Scientific) in staining buffer (2% FBS in HBSS (Corning)) for 30 minutes on ice and then washed twice with DPBS before cell sorting. Epidermal cells were sorted as E-cadherin (+) and α6-integrin (+). A431 tumors were collected from the mice and cut into small pieces (< 1mm3) with razor blades in 10cm plate. Chopped tumors were incubated with 0.25% collagenase (Millipore Sigma) in HBSS for 45 minutes. After collagenase incubation, chopped tumors were treated with 1 unit DNase (Millipore Sigma) for 10 minutes. Collect the tumors and medium filtered through 100μm filters and transfer the tumors from filters into 10cm plate. Tumors were incubated 0.25% Trypsin/EDTA (Corning) at 37C for 30 minutes. The A431 cells were stained with 1:200 Alexa fluor 700-β1-integrin (Biolegend), 1:200 APC-Cy7-α6-integrin (Biolegend), and 1:300 APC-CD11b, APC-CD31 and APC-CD45 (Biolegend) in staining buffer for 30 minutes on ice and then washed twice with DPBS before cell sorting. SCC cells were sorted as CD11b, CD31, CD45 (-), β1-integrin (high), and α6-integrin (high). Sequencing libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, NEB) and NEBNext Multiplex Oligos (NEB) for Illumina following the manufacturer's instructions. PCR-amplified libraries were purified using 1X ratio of Agencourt AMPure-XP Beads (Beckman) and eluted in 15μl TE buffer. All libraries were sequenced on an Illumina NextSeq using 50 bp paired-end reads.