GSM8113920: GG; Mus musculus; RNA-Seq
The whole colon (approximately 9-10 cm and weighted at circa 0.5 g) was freshly collected, dissected, washed, and cut open longitudinally, then placed it in cold DPBS (Gibco) with ice, rinsed for 5 times. Tissues were cut into 2-3 mm size with ophthalmic scissors, washed 15 times with DPBS. Biopsies were incubated in chelation medium (DPBS with 2 mM EDTA) at 37°C for 80 min with agitation. The supernatant was removed and replaced every 20 min, and the epithelial crypts contained supernatant was then digested into a single-cell suspension by dissociation with TrypLE Express (Gibco) containing 50 μg/ml DNase I (STEMCELL) for 1 h at 37°C. The epithelial single-cell suspension was washed and filtered with 40 μm cell strainers.  For further processing, cell counts and viability were confirmed with a Countess II automated cell counter (ThermoFisher Scientific) with confirmation by manual hemocytometer. In all single-cell and exploratory experiments, samples were processed immediately. Library was performed according to the manufacter's instructions (single ce 3' v2 protocol,10x Genomics). Firstly, single cell suspension should be obtained, and the single cell suspension should be tested for activity, and the cell activity is >90% before subsequent experiments can be carried out. To ensure the capture efficiency of the subsequent cells, the cell concentration is required to be controlled in the range of 700-1200cell/ul. During library preparation, Gel Bead and cells containing primer sequences linked to cell barcode and UMI (unique molecular identifier) are wrapped in "oil droplets" to form GEM (Gel Bead in Emulsion). Cells are lysed in this system and reverse transcribed to form full-length cDNA sequences. After the oil droplets are broken and purified, the cDNA library is amplified by PCR and sequencing primers are attached.