title: GSM4569729: pdgfraegfp_colon; Mus musculus; RNA-Seq
library_construction_protocol: Colonic tissue was harvested, minced into small pieces (2mm) and subsequently washed with PBS. Tissue pieces were then incubated in Gentle Cell Dissociation Reagent (STEMCELL Technologies) for 30 minutes at room temperature to detach the epithelium. The epithelial fraction was then filtered through a Falcon 70 µm cell strainer (Corning), washed with plain ADMEM/F12 and incubated for 5 minutes at 37 °C in prewarmed TrypLE express (Gibco), followed by single cell dissociation using the m_intestine program on the gentleMACS Octo Dissociator (Miltenyi Biotec). The epithelial single cell suspension was then filtered through a Falcon 40 µm cell strainer (Corning) and stored on ice in ADMEM/F12 (supplemented with 10% FBS). After the detachment of the epithelium the remaining tissue pieces were digested for 1 hour at 37°C under 110 rpm shaking conditions in DMEM supplemented with 2mg/mL collagenase D (Roche) and 0.4 mg/mL Dispase (Gibco). The mesenchymal fraction was then filtered through a Falcon 70 µm cell strainer (Corning), washed with plain ADMEM/F12 and subsequently filtered through a Falcon 40 µm cell strainer (Corning). Both epithelial and mesenchymal cells were stained for 30 minutes on ice with anti-CD45-PE (1:500, eBioscience) and anti-CD326(EpCAM)-FITC (1:500, eBioscience) in ADMEM/F12 (supplemented with 10% FBS). Prior to cell sorting, all cells were stained for 5 minutes on ice with Zombie Violet Fixable Viability Kit in PBS (1:1000, Biolegend). In the case of mesenchymal cell isolation from PdgfraH2BeGFP mice only staining with Zombie Violet Fixable Viability Kit was performed. Cells were detected and sorted at the Cytometry Facility at the University of Zürich using a FACSAria III cell sorter (Gates visible in corresponding Figures) (BD Biosciences). Library preparation was performed according to the manufacturer's indications (Chromium Next GEM Single Cell 3ʹ Reagent Kits v2 (PdgfraEGFP) or v3 (wt_colon) protocol)
attributes.tag: GEO Accession
attributes.value: GSM4569729