title: GSM6152646: Sample_1_Normal_pancreas_Flt3_YFPnegative_Macrophages; Mus musculus; RNA-Seq
library_construction_protocol: Mice were sacrificed by cardiac perfusion with ~10-15mL PBS-heparin under isofluorane anesthesia. Pancreas or tumor tissue was then isolated, minced with scissors, and digested in ~15-25mL Dulbecco's Modified Eagle Medium (DMEM) with 2mg/mL Collagenase A and 1x DNase I for either 15 minutes (steady-state pancreas and cerulein treated pancreas) or 25 minutes (tumor tissue) at 37°C with constant stirring. Digestion buffer was quenched with 3mL fetal bovine serum (FBS) and filtered through 100uM Nylon mesh, pelleted by centrifugation (2000rmp for 5 min) and resuspended in FACS buffer (1% BSA in PBS). Cells were then resuspended in FACS Buffer containing CD16/CD32 antibodies to block for 10 minutes on ice, pelleted by centrifugation, then resuspended in fluorophore-conjugated antibodies, stained for 25 minutes on ice, then washed with FACS Buffer. Cells were then immediately sorted on Aria-II (BD Biosciences).  For homeostatic pancreas, live macrophages (CD45+CD11b+CD3-CD19-B220-SiglecF-Ly6G-Ly6C-7AAD-F4/80+MHCIIHI/Lo) were sorted. For PDAC samples, live macrophages and DC enriched immune cells(CD45+CD3-CD19-SiglecF-Ly6G-7AAD-) were sorted. After cells were sorted into FACS Buffer, they were pelleted by centrifugation, resuspended in 0.04% BSA in PBS, and cell counts were obtained. For homeostatic pancreas, live macrophages (CD45+CD11b+CD3-CD19-B220-SiglecF-Ly6G-Ly6C-7AAD-F4/80+MHCIIHI/Lo) were sorted. For PDAC samples, live macrophages and DC enriched immunecells(CD45+CD3-CD19-SiglecF-Ly6G-7AAD-) were sorted. After cells were sorted into FACS Buffer, they were pelleted by centrifugation, resuspended in 0.04% BSA in PBS, and cell counts were obtained. For GEM/PTX and DMSO treated mice, live immune cells (CD45+EPCAM-CD31-) were sorted