title: GSM5194255: body skin; Gallus gallus; RNA-Seq
library_construction_protocol: One day old chicks (strain Lohmann) were obtained from Schropper GmbH, Gloggnitz, Austria. Skin was excised from the leg of sacrificed animals and incubated in thermolysin (0.5 mg/ml) (Sigma-Aldrich, #T7902) at 37°C for 45 minutes. The lower dermis was removed using forceps and the remaining tissue, including the epidermis and parts of the upper dermis, was processed further according to protocols established for human skin (Vorstandlechner et al. 2020; Kalinina et al. 2020). For the isolation of cells, the tissue was split into two fractions that were incubated either in buffer-enzyme mix of the Whole Skin Dissociation Kit human (MACS Milteny Biotech, #130-101-540) for 2.5 h at 37°C or with 0.05% trypsin/EDTA (Thermo Fisher Scientific, #25300) and DNase 1 (10 µg/ml) (Roche Diagnostics, #10104159001) at 37°C for 15 minutes. Afterwards the samples were combined and processed according to the manufactor's protocol (Whole Skin Dissociation Kit human, MACS Milteny Biotech, #130-101-540). In brief, the epidermis-enzyme mix was diluted in 0.5 ml medium and dissociated with the gentleMACS Dissociator. The ground tissue was filtered through 100-micron (Falcon, #352360) and 40-micron (Falcon, # 352340) meshes. Subsequently, cells were stained with DAPI and viable cells were purified by fluorescence-activated cell sorting at the Core Facility Flow Cytometry, Medical University of Vienna. Sequencing libraries of viable cells were prepared using Cell Ranger (10x Genomics) version 3.0.2 at the Center for Molecular Medicine, Vienna, Austria. 10x Genomics Chromium Single Cell 3' Reagent Kit v2