GSM6509085: NeuroD1ST_1 [RNA-seq]; Mus musculus; RNA-Seq
The pancreata microdissected from E15.5 embryos were directly trypsinized and prepared for FACS. Briefly, tissue was dissociated using 0.05% trypsin/0.53 mM EDTA at 37°C for 5 min. Digestion was stopped by adding the FACS buffer (2% FBS and 10 mM EGTA in PBS), and cells were then 1x washed by FACS buffer. Finally, cell suspensions were filtered through 40 μm nylon mesh and immediately tdTomato+ cells were sorted using a flow cytometer (BD FACSAriaTM Fusion), through a 100 μm nozzle in 20 psi, operated with BD FACSDiva™ Software. 100 sorted cells were collected into the 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). The plates were frozen immediately on dry ice and stored at −80°C until the libraries were prepared following the manufacturer's instructions of previously mentioned NEB Next kit.