scRNA-seq of homo sapiens: Bowen's disease
For scRNA-seq, 3mm punch biopsies were obtained from fresh skin at different stages of the cSCC disease continuum. Fresh human skin tissue was kept in MACS Tissue Storage Solution (Miltenyi Biotec, cat. no. 130-100-008) at most overnight. It was then enzymatically and mechanically dissociated using the Whole Skin Dissociation kit for human material (Miltenyi Biotec, cat. no. 130-101-540) and the Gentle MACS Dissociator device (Miltenyi Biotec), following the manufacturer's instructions. The resulting cell suspensions were filtered through 70m cell strainers (Falcon, cat. no. 10788201) and the Dead Cell Removal Kit (Miltenyi Biotec, cat. no. 130-090-101) was then used to remove apoptotic and dead cells. Subsequently, about 20,000 single cells per sample were used to prepare sequencing libraries with the Chromium Single Cell Reagent kit (v3.1 chemistry) from 10X Genomics (cat. no. 1000128), following the manufacturer's instructions. Library quality was checked with the Qubit dsDNA HS Assay kit (Life Technologies, cat. no. Q32851), and cDNA integrity was assessed with D1000 ScreenTapes (Agilent Technologies, cat. no. 5067-5582). Finally, pairwise sequencing (26 + 96bp) was performed with a NovaSeq 6000 device (Illumina).