GSM8389760: Forestomach, DEN, Batch2, Rep1; Mus musculus; RNA-Seq
For scRNA-Seq, Fucci2a male mice were sacrificed 8 months after DEN treatment (age-matched mice were used as controls). After dissection as described above, tissues were flattened with the epithelial side up and visible tumour lesions were marked by making a half-cut using a 1 mm diameter punch under the dissecting microscope. Then, oesophagi and forestomachs were cut in half and incubated in 50 mM EDTA in PBS containing 1 U/µl RNAse Inhibitor (Life Technologies; AM2696) at 37°C for 30 minutes. Following incubation, the epithelium was carefully peeled away from the stroma using fine forceps. Epithelium and stroma were each flattened individually and tumours labelled with the half incision described above were then fully excised using 0.5-2 mm punch biopsies. Pools of biopsies from two to four animals (details below) were further processed as follows. Stroma was thoroughly minced and incubated in 4 mg/ml collagenase type 4 (Thermo Fisher Scientific; 17104019) in DMEM with 1 U/ml RNAse inhibitor for 1 h at 37°C in a rotator. EDTA was added to the samples at a final concentration of 5 mM and the suspension was diluted 1:5 in collection buffer (2% heat-inactivated fetal bovine serum (Life Technologies, 26140079), 25 mM HEPES (Life Technologies, 15630056) in PBS to reduce the collagenase activity. Cell suspensions were filtered through a 100 µm cell strainer, centrifuged at 300 × g for 5 min at 4°C, and resuspended in collection buffer before an additional filtering step with a 40 µm FlowMi tip filter (Thermo Fisher Scientific, 15342931) was performed to obtain single-cell suspensions. Cells were then centrifuged at 300 × g for 5 min at 4°C and resuspended in collection buffer containing 1 U/µl RNAse. In parallel, the epithelium was thoroughly minced and incubated for 10 min at 37°C in 0.5 mg/ml dispase (Sigma; D4818) in PBS containing 1 U/µl RNAse to create an epithelial cell suspension. EDTA was then added to the samples at a final concentration of 5 mM and the suspension diluted 1:5 in collection buffer to reduce the dispase activity. A single-cell suspension was obtained by filtering the samples through a 30 µm cell strainer. Cells were then centrifuged at 300 × g for 10 min at 4°C and resuspended in collection buffer containing 1 U/µl RNAse. Epithelial and stromal cells were then pooled together by condition (Ctrl, DEN, Tumour) prior to proceeding to library production for scRNA-seq. Single-cell RNA-seq libraries were generated using the 10x Genomics Chromium Next GEM Single Cell 3' Reagent Kit (v3) and sequenced at the Genomics Core Facility of Cancer Research UK (CRUK), Cambridge Institute following the manufacturer's instructions