GSM3938451: Control Embryonic Day 13.5; Mus musculus; RNA-Seq
At noon, embryos were harvested by hysterectomy and the pancreas was dissected under a microscope in ice-cold phosphate buffered saline (PBS) and transferred into 2 mL of prewarmed 0.25% Trypsin-EDTA (TE) in a 15 mL Falcon tube. Falcon tubes were placed in a 37°C bead bath for 8 minutes of digestion, followed by addition of 1 mL of FBS and 2 mL of PBS to stop the digestion. The cell solution was passed through a 40 µM cell strainer into a new 15 mL Falcon tube. Single cells were spun at 300xg for 5 minutes and all but ~50 µL of supernatant was removed. For FACS sorting, 300 µL of 10% FBS in PBS was added to the cells, transferred to a FACS tube where 7-AAD was added to stain dead cells, and ~30,000 live single cells from each embryo were sorted into 1 mL of 10% FBS in PBS. Cell counts and viability were confirmed under the microscope on a hemocytometer using Trypan Blue and cell numbers were estimated to be ~300 cells/µL. Using a cell concentration of 300 cells/µL, 29 µL of cells were loaded for a targeted recovery of 5,000 cells according to the Chromium Single Cell 3' Reagent Kits v2 User Guide (10X Genomics, Inc.), which was followed as described. Single Cell RNA-seq