GSM8458233: non_IR_2; Mus musculus; RNA-Seq
Colonic epithelial cells and stromal cells were isolated separately. For the isolation of colonic epithelial cells, the colon was extracted, washed, longitudinally cut, and incubated in 2 mM EDTA in PBS at 4°C for 20 minutes. Colonic crypts were isolated by direct scraping using slides and enriched through centrifugation. Subsequently, the colonic crypts were digested in Collagenase I (2 mg/mL, Sigma-Aldrich) at 37°C for 30 minutes. Single cells were obtained by filtering through a 40 μm cell strainer (BD Biosciences). The cells were then centrifuged and resuspended in Advanced DMEM/F12 (Gibco, 12634010) with Propidium Iodide (PI, 5 μg/mL). PI-negative live cells were isolated by flow cytometry (MoFlo Astrios EQ).For the isolation of colonic stromal cells, colon was washed with cold PBS first and then incubated in cold PBS containing DTT (1 mM) at 250 rpm for 10 min to remove chymus. Next, colon was transferred to cold PBS containing EDTA (50 mM) and HEPES (10 mM) and colonic epithelium was removed by vigorous shaking for 2 min. The last step was repeated to fully remove the epithelium. The remaining tissue was incubated in RPMI1640 containing Collagenase IV (1000 U/mL, Sigma-Aldrich) at 37°C for 60 min. The digested tissue was minced through pipetting and the muscle tissue was removed through filtering by a 70 mm cell strainer (BD Biosciences). Stroma cells were centrifuged and resuspended in MEM (Gibco, 12571063) and live stromal cells were also isolated by flow cytometry. To conduct single-cell RNA-sequencing, Chromium Single Cell 3ʹ Reagent Kits v3 were chosen (10x Genomics). Single live colonic epithelial cells and stromal cells were isolated from mice at the designated time point after irradiation. For each sample, epithelial cells and stromal cells were mixed at a ratio of 1:1 before loading to the Chromium controller (10x Genomics). Reverse transcription, cDNA amplification, and library preparation were completed according to the manufacturer's recommendations. scRNA libraries were sequenced using HiSeq X Ten system (PE-150 bp, Illumina).