title: GSM8241649: Colon, rockroi unimodal; Mus musculus; OTHER
library_construction_protocol: Murine colonic tissues from Pdgfra-H2B-EGFP reporter mice (Hamilton et al., 2003) were flushed with PBS and finely minced into 2 mm pieces. Minced tissue fragments were washed three times with PBS. For the detachment of the epithelial fraction, the tissue pieces were incubated in Gentle Cell Dissociation Reagent (STEMCELL Technologies, Germany) while gently rocking for 30 minutes at room temperature. The pieces were pipetted up and down for the epithelial fraction to be detached. The epithelial fraction was filtered through a Falcon 70-μm cell strainer (Corning, Switzerland), washed with plain ADMEM/F12 and incubated for 5 minutes at 37°C in prewarmed TrypLE express (Gibco, Thermofisher, Switzerland). The gentleMACS Octo Dissociator (Miltenyi Biotec, Switzerland) m_intestine program was employed for single-cell dissociation. The obtained epithelial single-cell suspension was filtered through a Falcon 40-μm cell strainer (Corning) and kept on ice in ADMEM/F12 supplemented with 10% FBS. For dissociation of the mesenchymal fraction, the remaining tissue pieces (following epithelium detachment) were digested for 1 hour at 37°C under 110 revolutions per minute (rpm) shaking conditions in DMEM supplemented with 2 mg/mL collagenase D (Roche) and 0.4 mg/mL Dispase (Gibco). The mesenchymal fraction was 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). The epithelial and mesenchymal cells were mixed and stained for 30 minutes on ice with anti-CD326(EpCAM)-PE-Cy5 (1:500, eBioscience/Thermofisher, Switzerland) in PBS. Prior to cell sorting, all cells were stained for 5 minutes on ice with DAPI in PBS (1:1000, ThermoFisher, Switzerland). Epithelial and mesenchymal cells labeled with PE-Cy5 and EGFP were sorted separately and subsequently mixed in a 1:1 ratio. Cells were sorted at the Cytometry Facility at the University of Zürich using a FACSAria III cell sorter (BD Biosciences, Switzerland). Libraries were generated on a BD Rhapsody Express machine. Libraries for unmodified samples were generated following manufacturers recommendations for the WTA assay. The protocol for v1 BD Rhapsody was used (i.e. a single RPE reaction) and applied to enhanced beads. The RoCK and ROI sample was generated following the RoCK and ROI protocol. Briefly, the protocol differs from the standard WTA protocol due to the following points: 1) addtion of N1 primer to the RPE PCR; 2) addition of ROIseq primers to the pool of randomeres during RPE extension; 3) separate indexing reaction for the samples in which the N1 primer is added; 4) custom sequencing primer