WGS of patient 13 PDAC tissue: Sample P13_T_WGS
"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. Tumor DNA was extracted from tissue sample using Qiagen Genomic-tips and Qiagen blood and cell culture DNA mini kit. Briefly, tissue sample was minced and homogenized mechanically using Qiagen TissueRuptor II in lysis buffer, supplemented with Qiagen Proteinase K and incubated overnight at 50 oC. Thereafter, genomic DNA was purified following QIAGEN Genomic-tips procedure according to the manufacturer's instructions. DNA yield was determined fluorometrically using the High Sensitivity dsDNA kit (Invitrogen) on Qubit 3.0 Fluorometer and DNA purity was estimated spectrophotometrically by measuring absorbance on QuickDrop (Molecular Devices).Sequencing libraries were generated by Novogene with library size of 350 bp."