GSM8150946: Progressing papilloma (early carcinoma) 5, scRNAseq; Mus musculus; RNA-Seq
Single-cell suspensions of dorsal back skin were prepared by carefully removing subcutaneous fat and floating the intact tissue dermal side down in 0.25% Trypsin/EDTA (Invitrogen, 25200-056) solution with gentle rocking for 1 h at 37°C. Trypsin activity was neutralized with 10% chelexed FBS in Ca2+ free PBS (Hyclone, SH30028.02). The epithelial skin layer was then scraped into suspension and agitated to dissociate cells. To obtain single-cell suspensions of skin tumors, lesions were finely chopped into very small pieces with a sterile scalpel and then enzymatically digested in 0.25% Trypsin-EDTA with gentle agitation for 1 hour at 37°C. Individually, each disaggregated cell suspension sample was then passed through a sterile 40 µm filter, centrifuged at 300 g for 5 min to pellet cells, and resuspended in 10% chelexed FBS in Ca2+ free PBS prior to flow cytometry. Cell samples were collected and analyzed using a FACSAria III, and appropriate gating was used to exclude doublets. DAPI staining was used to assess cell viability, and only viable, DAPI-negative cells were selected for subsequent analysis. Within each sample type, three populations of viable cells were evaluated: unsorted global cell populations, Lgr6eGFP+ stem cells (gated on positive eGFP expression), and tdTomato+ Lgr6+ stem cell-derived progeny cells (gated on positive tdTomato expression). FACS sorted cells were collected in Ca2+ free, PBS-containing 1% chelexed Ca2+ free FBS, centrifuged at 300 g to pellet the cells before final resuspension in 35 µl 1% chelexed Ca2+ free PBS, and stored on ice prior to scRNA-sequencing library preparation which occurred within 2-3 h of sample collection. scRNAseq libraries from samples were prepared using the 10x Genomics Chromium extraction and preparation protocol according to the manufacturer's instructions and sequenced on a Novaseq S4 sequencer (Illumina).