GSM9461701: pGF-DSS-7; Mus musculus; RNA-Seq
The intestines of mice were dissected and washed with pre-cooled PBS to remove feces and mucus until the cleaning solution became clear. Subsequently, the intestines were sectioned into 0.5 cm segments, transferred into preheated epithelial cell removal solution (PBS with 10 mM EDTA), and incubated at 37 °C, 30 rpm for 15 min on a rotating shaker. The tissues were then incubated on ice for 15 min, and then washed twice with PBS containing 2 % fetal bovine serum (FBS). The remaining tissues were placed in a preheated digestive solution [RPMI-1640, 100 μg/ml Liberase TL (5401020001, Roche, Switzerland), and 50 μg/ml DNase I (11284932001, Roche, Switzerland)] at 37 °C, 40 rpm for 30 min on a rotating shaker. The supernatant was collected, and pre-cooled MACS buffer (PBS, 2 % FBS, and 2 mM EDTA) was added to terminate digestion.After washing with MACS buffer, cells were resuspended in MACS buffer containing 0.125 μg/100 μl fluorescein-coupled CD45 monoclonal antibody (12-0451-82, Thermo Fisher, USA) at a concentration of 2×106 cells/100 μl. After incubation at room temperature for 30 min, the unlabeled antibody was washed away. The cells were re-suspended in MACS buffer for flow sorting. CD45-positive cells were collected in a collection tube containing RPMI-1640 and 5 % FBS. ScRNA-seq was performed using Chromium Single Cell 5' Gel Bead and Library Construction Kit (10×Genomics, USA) and Single Cell V(D)J Amplification Kit (10×Genomics, USA). Reverse transcription, cDNA recovery, cDNA amplification, and library construction were performed according to the manufacturer's protocol.