GSM6046166: fibroblast_normal_day4rep1; Mus musculus; RNA-Seq
GSM6046166
Normal pancreas: Dissociation media for normal pancreas is Collagenase P (1 mg/ml), Soybean Trypsin inhibitor (0.2 mg/ml), Dnase I (12 U/ml), HEPES (10 mM), Rock inhibitor Y (10 μM) in HBSS (with Ca2+). Pancreas was dissected, minced in Eppendorf tubes, and then transferred to a Miltenyi gentleMACS tube with 2.5ml of dissociation media. Dissociation was performed on a gentleMACS Dissociator using program 37m-TDK-1 for 15 min. For tumor-bearing pancreata: Dissociation media for tumor bearing pancreas was prepared using the Tumor Dissociation Kit (Miltenyi Biotec, 130-096-730). Pancreas was dissected and cut into small pieces using a razor blade. A full program of 37m-TDK-1 was used to dissociate the tumor. Similar procedures were then used for both normal and tumor pancreata. 15ml of cold DMEM+10% fetal bovine serum (FBS) dissociation mix was then filtered through a 70 μm and then a 100 μm membrane. Red blood cell lysis buffer was then added to the pellate and pipetted. After centrifuge, cells are suspended in FACS buffer (5% FBS, 10 μM Y, 20 mM HEPES, 5 mM MgCl2, 12 U/ml DNase I in PBS). After Fc block, cells were incubated with fluorophore conjugated antibodies (Extended Data Table 2). A BD FACS Aria machine was used to ether analyze or sort the cells.   Tumor pancreas: Dissociation media for tumor bearing pancreas was prepared using the Miltenyi gentleMACS tumor dissociation kit. Pancreas was dissected and cut into small pieces using a razor blade. A full program of 37m-TDK-1 was used to dissociate the tumor. Procedures after dissociation were the same as the normal pancreas. After dissociation and staining, cells were sorted into three groups, Group 1. hematopoietic cells; Group 2. endothelial + epithelial cells; and Group 3. fibroblasts. A fraction of Group 1 cells was combined to Group 2. The fraction was calculated to achieve similar cell numbers of Group 1 and Group 2. For tumor samples, 4-5 mice were pooled to generate each replicate (n=2). For normal samples, 6 mice were pooled, sorted and split into 2 replicates (n=2).  Cells were resuspended in PBS and counted with trypan blue to evaluate viability.  To create a pool of hematopoietic and endothelial + epithelial cells, an equal ratio of cells were mixed from these two groups to establish the pool 1+2.  Library preparation and sequencing were completed at MCW's Genomic Science and Precision Medicine Center with 10,000 cells aliquoted for groups 1+2 and 3, and were processed according to the Chromium Next GEM Single Cell 3' Reagent Kits (10X Genomics, dual indexes). All samples passed quality control metrics with 13-16 cycles of PCR completed to amplify the libraries.  Final libraries were quantified and pooled by qPCR (Kapa Library Quantification Kit, Kapa Biosystems) and sequencing completed on the NovaSeq6000 (GSPMC at MCW) targeting 50,000 reads per cell per condition.