GSM8979113: Control_Small_Intestine_Mouse; Mus musculus; RNA-Seq
The mouse colon or small intestine were dissected and washed with HBSS (Gibco) supplemented with antibiotic antimycotic solution (Gibco). Epithelial cells were removed by incubating the tissue with 5 mM EDTA and 1 mM DTT in HBSS for 25 minutes at 37°C. The tissues were digested in 300 U/mL Collagenase XI (Sigma-Aldrich) and 1 mg/mL Dispase II (Roche) in DMEM at 37°C for 60 min or 45 min, for the colon and small intestine, respectively. Cell suspensions were centrifuged and washed in FACS buffer (5% FBS (Biowest) in PBS). 1–2 million cells/100 μL were incubated with anti-CD45 and anti-CD326 antibodies for the exclusion of immune and remaining epithelial cells, respectively, and 1 μg/mL Propidium Iodide (PI) (Sigma) for dead cell exclusion. Cell sorting was performed using the FACSAria III cell sorter (BD) and the FACSDiva (BD) software. For each sample, approximately 16,000 cells were loaded onto a 10X Genomics Chromium Controller using the Chromium Next GEM Single Cell 3' Kit v3.1 according to the manufacturer's protocol. Library preparation was performed following the 10X Genomics workflow, with quality control performed using an Agilent Bioanalyzer High Sensitivity DNA chip.