scRNA-seq of patient 13 PDAC tissue: Sample P13_T_SC
"Tumor tissue was minced and subsequently dissociated by enzymatic digestion (200 units/ml Collagenase type I, 60 units/ml hyaluronidase, and 100 ug/ml DNAse I, dissolved in DMEM media without serum) for 30 min at 37oC. Single cell suspensions were made by passing the digested tissue through a 100 um filter.  Recovered cells were centrifuged at 500xg for 5 minutes and washed with Phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA). Washed cells were pelleted.  To eliminate red blood cells (RBC), cell pellets were resuspended in ACK lysing buffer (Thermofisher/Gibco) for 5 minutes at room temperature, and then pelleted. Cells were again washed in PBS/0.5% BSA, pelleted, and finally resuspended in PBS/0.5% BSA. For FACS isolation of CA19+ cells, cells were treated with a rabbit monoclonal antibody against CA19 (Novus NBP2-54585), which was conjugated to APC. The antibody treatment was for 30 minutes at room temperature, and then cells were pelleted by microcentrifugation at 500xg for 5 minutes. After washing in PBS/0.5%BSA and repelleting, the cells were resuspended in PBS/0.5%BSA. DAPI was added to indicate viability. Standard illumina protocols were used to prepare scRNA-sea libraries."