GSM9635075: intestinal epithelial cells, 25-week-old, TrnA G5081A point mutant, 81% mutation load, male; Mus musculus; RNA-Seq
Mouse small intestines were excised and flushed with cold PBS. The tissues were opened longitudinally along the lumen and cut into 1 cm segments. The segments were incubated in cold PBS containing 20 mM EDTA for 1 hour on ice. Following incubation, the supernatant was discarded and replaced with fresh cold PBS. The tubes were then vortexed for 30 seconds to dissociate villi and crypts from the basement membrane. The released villi and crypts were transferred to a new tube and digested with TrypLE (Thermo Fisher Scientific, 12604013) supplemented with 10 mg/mL DNase I (Roche, 10104159001) for 10 minutes at 32°C. The digested cell suspension was sequentially filtered through 70-μm and 40-μm cell strainers. Isolated single IECs were centrifuged and resuspended in cold Dulbecco's Phosphate-Buffered Saline (DPBS) containing 2% fetal bovine serum (FBS). For flow cytometry sorting, cell suspensions were stained on ice for 30 minutes with the following antibodies diluted in cold 2% FBS in DPBS: CD45-APC (1:100, clone 30-F11, BioLegend, 103112), CD31-PE (1:50, clone MEC13.3, BioLegend, 102507), TER-119-Brilliant Violet 510™ (1:50, clone TER-119, BioLegend, 116237), and CD326-APC/Cyanine7 (1:100, clone G8.8, BioLegend, 118217). Following incubation, cells were washed with cold 2% FBS in DPBS, resuspended in the same buffer supplemented with 0.1 μg/mL DAPI (BioLegend, 422801), and filtered through a 40-μm cell strainer. Fluorescence-activated cell sorting was performed using a BD FACSAria™ Fusion instrument, and the sorting strategy was visualized using FlowJo Software. The scRNA-seq was performed using the 10× Chromium Next GEM Automated Single Cell 3′ Reagent Kits v3.1 (1000141) following the manufacturer's protocol (CG000286 • Rev F). Sorted cells were loaded onto a Chromium Next GEM Automated Chip G (1000136), and single-cell gel bead-in-emulsions (GEMs) were generated using the Chromium Controller Single-Cell Instrument. Following reverse transcription and library construction, library quality was assessed using the Qubit dsDNA HS Assay Kit (Invitrogen) and the High Sensitivity DNA chip (Agilent). Sequencing was performed on the NovaSeq X Plus platform using a 2 × 150 bp paired-end read configuration.