GSM8248141: SH, scRNAseq; Mus musculus; RNA-Seq
The small intestines were carefully cleaned and opened longitudinally, washed with PBS, cut into 1.0–1.5 cm pieces and incubated in 20 ml of HBSS/10 mM HEPES, 8% FBS, 4 mM EDTA, and 0.5 mM dithiothreitol at 37 ℃ with constant agitation for 15 minutes twice. After washing with Ca2+ /Mg2+ -PBS to remove EDTA, the remaining pieces of small intestines were minced and digested in RPMI-1640 medium containing fetal bovine serum (FBS), collagenase Ⅳ (0.1 mg/mL) and DNase I (0.1 mg/mL) at 37 ℃ for 40 minutes. The tissue digestion was filtered through a 40 μm cell strainer, following by centrifugation at 400g for 5 min at 4 °C. CD45+ Cells were collected using FACS, and resuspended in PBS. Cell concentration was adjusted to 1000 cells /μl for library preparation. Prepared single-cell suspensions were loaded on a chip of the Single Cell 3' Chip Kit (10X Genomics) and subjected to a Chromium Single Cell Instrument (10X Genomics) to generate single-cell gel beads in emulsions (GEMs). cDNA amplification and library construction underwent using a Chromium Single Cell 3' Reagent Kits v2 (10x Genomics).