GSM7719805: pancreatic tumor, 10952_S3; Mus musculus; RNA-Seq
All mouse pancreatic tumors were aseptically taken out from tumor-bearing mice, washed with cold 1x PBS (GIBCO, Life Technologies), and minced into 1-2 mm pieces using scalpel blades immediately following resection. Tumor pieces were collected into a 50 ml Falcon tube containing 10 mL Gey's balanced salt solution (GBSS, Sigma, #G9779), 5 mg Collagenase P (Roche, #11249002001), and 0.2 µg DNase I (Roche, #4716728001). Samples were incubated for 20 to 30 minutes at 37oC, then pipetted and returned to 37oC for 15 minutes. Samples were re-pipetted then passed through a 70 µm nylon mesh (Corning). Red blood cells were lysed using RBC lysis buffer (eBioscience, #00-4333-57), and remaining tumor cells were washed, resuspended in GBSS (GIBCO, Life Technologies) containing 2.5% FBS and 2 mM EDTA for staining, FACS analysis, and cell sorting. Sorted cells were washed once and resuspended in 1x PBS with 0.05% BSA. Sorted cells were manually counted and viability was assessed using Trypan Blue staining. Up to 16,000 cells were loaded into one well of the 10x chip to produce Gel Bead-in-Emulsions (GEMs) and capture individual cells. We utilized the Chromium Single Cell 3' Reagent Version 1 Kit (10X Genomics).  GEMs underwent reverse transcription to barcode RNA before cleanup and cDNA amplification. Libraries were prepared with the Chromium Single Cell 3' Reagent Version 3 Kit. Each sample was sequenced on HiSeq4000 (Illumina) in Rapid Run Mode with paired-end sequencing parameters for an average depth of 50,000 reads per cell.