Single cell RNA sequencing of mice skin cells in DMBA/TPA-induced tumorigenesis model

WT or Cd226-/- mice were treated with DMBA and TPA. Three weeks later, cells were isolated from mice back skin, and divided CD45+ and CD45- cells by cell sorter. CD45+ and CD45- cells were mixed in 1:1 ratio, and we performed scRNA sequencing of skin cells. Overall design: WT or Cd226-/- mice on the BALB/c background were treated with DMBA and TPA. Three weeks after DMBA/TPA-treatment, cells were isolated from mice back skin. For each sample, skin cells were pooled from three mice. Skin cells were stained with biotin conjugated anti-mouse CD45.2 mAb, and then stained with barcode sequence-conjugated PE Streptavidin (TotalSeq™-A0951, BioLegend). CD45+ and CD45- cells were sorted by cell soter, and mixed in 1:1 ratio. For naive mice sample, CD45+ cells from WT and Cd226-/- mice were pooled, CD45- cells from WT and Cd226-/- mice were pooled, in 1:1 ratio. Viability of all samples were more than 70%. Single cells were processed through the Chromium Single Cell Platform using the Chromium Single Cell 3' Library and Gel Bead Kit v3.1 (10X Genomics, PN-1000128), and Chromium Next GEM Chip G Single Cell Kit (PN-1000127, 10X Genomics), and the TotalSeq™-A Antibodies with 10x Single Cell 3' Reagent Kit v3.1 (BioLegend) as per the manufacturer's protocol. The sequencing of libraries was performed at Macrogen Japan (Kyoto, Japan) using an Illumina HiSeq X. Processed files were generated from Cell Ranger (10X Genomics) pipeline.