GSM4661656: E14.5; Mus musculus; RNA-Seq
Adult 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). Embryonic hindguts isolated and pooled from each stages including E14.5 (25 embryos), E15.5 (17 embryos) and E18.5 (14 embryos). Tissues were minced into small pieces and then incubated for 60 min @ 37 °C in PBS with 15mg of collagenase  in 100ml. Single cells were centrifuged and resuspended in PBS+ 0.2% BSA. Then cells were subjected to antibody staining prior sorting with EpCAM-TITC (eBioscience) and CD45-PE (eBioscience) for 45 minutes. After washing and resuspension in PBS, samples were incubated with Zombie Violet Fixable Viability Kit in PBS (Biolegend) for 5 minutes on ice. Single cells were then centrifuged, resuspended in BGJb media+ 10% FBS and filtered through Falcon 40 µm cell strainer (Corning) and kept on ice prior sorting. Cells were detected and sorted at the Cytometry Facility at the University of Zürich using a FACSAria III cell sorter (BD Biosciences). Library preparation was performed according to the manufacturer's indications (Chromium Next GEM Single Cell 3ʹ Reagent Kits  v3  protocol)
GEO Accession
GSM4661656