scRNA_E130ESO_Stroma
Esophageal tubes were cut open using fine scissors, and gently digested in 6 cm dishes with 25 U mL Dispase (Corning) + 100 μg mL DNase I (Millipore Sigma) at room temperature for 20 to 40 min. Digestion was stopped by transferring esophagus to cold HBSS. Epithelium was physically peeled off from surrounding stromal tissues with fine forceps, and collected in a 15 ml Falcon tube. Stromal tissues were transferred to clean dishes and chopped into small pieces using scissors. Tissue pieces were transferred to a 15 ml Falcon tube with 10 ml collagenase solution (5 mg mL Collagenase in DKSFM), triturated gently using P1000 pipette, and then shaken in a Thermomixer at 37 ​​°C at 1000 rpm for 10 to 30 min. 1 ml 0.25% Trypsin was then added to the solution for an additional 5 min dissociation. Dissociation was stopped by addition of the FACS buffer supplemented with 10% FBS. Dissociated cells were resuspended in cold 0.04% BSA in PBS and filtered through 40 μm strainers. Cells were transferred to 1.5 ml Eppendorf tubes and pelleted down at 300 g at 4 °C for 5 min. Then cells were resuspended in cold 0.04% BSA in PBS for cell number counting. Cell alive rate was determined using Trypan Blue. Only samples with greater than 80% cell alive rate were further processed. Cells were then subjected to 10X Chromium scRNA-Seq based on the manual.