GSM9092863: GF_bulk_3; Mus musculus; RNA-Seq
To obtain all cell types from the intestine for single-cell RNA-seq, we collected intestine segments from GF and SPF male and female C57BL6/J mouse pairs in ice-cold 2% FBS/PBS. After removing fat tissue, segments were cut open longitudinally and washed with ice-cold PBS. Epithelial cells were stripped with a stripping buffer (5 mM EDTA, 1 mM DTT, 2.5 mM HEPES, and 5% FBS in HBSS) for 30 min at 37°C with stirring. Tissues were collected and rinsed with ice-cold 2% FBS/PBS, followed by enzymatic digestion with 5 mL digestion buffer (0.2 mg/mL collagenase IV, 0.2 mg/mL dispase II, 0.1 mg/mL DNase I in DMEM/F12) at 40 °C for 10 min. Digested tissues were allowed to rest for 5 min, and the supernatants were collected into ice-cold complete media (CO2 independent media,18045088​​, Thermo Fisher Scientific; with 2% FBS) with 10 mM EDTA and kept on ice. This process was repeated for approximately 45 min with gentle vortex until no tissue chunks remained. Cells were pooled together and filtered through a 70 μm cell strainer, centrifuged at 500g at 4°C for 10 min. Cell pellets were resuspended in complete media (CO2 independent media with 2% FBS) with 10 mM EDTA. 150uL of the cell suspension was immediately loaded on a 10x controller as "bulk", while the remaining 850uL were sorted to enrich for stromal cells by excluding cells positive for Epcam, Ter119 and CD45 using a MoFlo Astrios sorter. The sorted negative cells were then loaded on a 10x controller as "stroma". Single cell suspensions were processed using the Chromium Single Cell 3' Gene Expression kit (v2, 10x Genomics) per manufacturer's instructions.