GSM8964404: Epithelial Cells, Pregnant; Mus musculus; RNA-Seq
Small intestines were collected from CO2 euthanized animals and the first 12 cm was sectioned. The tissues were opened longitudinally, washed two times with 5% gut wash buffer (HBSS supplemented with 25 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 mM nonessential amino acids, 100 U/ml penicillin and 100 mg/ml streptomycin with 5% FBS) and cut into 2-5 mm pieces into 10 ml of gut wash buffer containing 2mM DTT. The tissue was incubated in a 37°C water bath for 10 min, briefly shaken every 2 minutes, and passed through a 100 μm filter after vigorous shaking 15 times. The tissue was then transferred to a new conical containing 10 ml of gut wash buffer with 5mM EDTA and incubated at 37°C for 8 minutes with brief shaking every 2 minutes. The supernatant fraction was collected on ice by passing through a 100 μm filter and the tissue was transferred to a new conical to repeat the EDTA wash step for a total of three times. The second and third fractions were collected in the same tube as the first one. Cells were centrifuged at 1000 rpm for 5 min at 4°C and washed once with 0% DMEM. The cell pellet was incubated in 10 ml of DMEM supplemented with 0.1 U/ml Dispase and 0.1 mg/ml DNase in a 37°C water bath for 8 minutes, with brief shaking every 2 minutes. After neutralization with 5 ml 2.5% DMEM, the cells were centrifuged at 1000 rpm for 5 min and washed once with the staining buffer (same as 5% gut wash buffer). Antibodies diluted in the staining buffer were added and staining was performed on ice for 20 minutes. The cells were washed with 10 volumes of DMEM and resuspended in 10% DMEM for sorting. All cell sorting was performed on Sony MA900 sorter. Prior to loading onto the sorter, cell suspension was filtered using a 5 ml falcon tube with a cell strainer cap and DAPI was added with a 1:400 concentration. Alive EPCAM+ (Anti-EpCAM (AF594; 1:1000; G8.8; Biolegend)) epithelial cells were sorted directly into 10% DMEM. Libraries were prepared using Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomics) following manufacturer's instructions