Pancreatic Endocrine Cell Isolation and Sorting

The pancreases from P9-P10 pups were first perfused by 1 mg/ml collagenase in Hank’s balanced salt solution, dissected, and incubated at 37 °C for 10–12 min to release endocrine cells/islets. Digested pancreatic tissue was washed 3 × by 1% FBS in Hank’s solution. To generate single cells, the tissue was further dissociated by trypsinization as described [66 (link)]. Briefly, tissue was dissociated using 0.05% trypsin/0.53 mM EDTA at 37 °C for 5 min. Digestion was stopped by the FACS buffer (2% FBS and 10 mM EGTA in PBS [66 (link)]), and cells were then 1 × washed by FACS buffer. The pancreases microdissected from E14.5 embryos were directly trypsinized and prepared for FACS as described above. Finally, cell suspensions were filtered through 40 µm nylon mesh and immediately tdTomato⁺ cells were sorted using a flow cytometer (BD FACSAria™ Fusion), through a 100 µm nozzle in 20 psi, operated with BD FACSDiva™ Software (Additional file 1: Fig. S7). For RNA sequencing, 100 sorted cells were collected into individual wells of 96-well plate containing 5 µl of lysis buffer of NEB Next single-cell low input RNA library prep kit for Illumina (New England Biolabs #E6420). Plates were frozen immediately on dry ice and stored at − 80 °C. The total time from euthanasia to cell collection was ∼3 h. For the epigenetic study, on average, 4700 cells/sample at E14.5 and 14,700 cells/sample at P9 were sorted. Cell sorting was performed in the Imaging Methods Core Facility at BIOCEV.