After all regions of interest were dissected, the ACSF.I was removed and 1 ml of a 2 mg/ml pronase in ACSF.I solution was added. Tissue was digested at room temperature (approximately 22°C) for a duration that consisted of adding 15 minutes to the age of the mouse (in days; i.e., P53 specimen had a digestion time of 68 minutes). After digestion, the pronase solution was removed and replaced by 1 ml of ACSF.I supplemented to a concentration of 1% FBS (Fetal Bovine Serum). The tissue was washed two more times with the same solution with the third wash being 500 μl for final sample volume. The sample was then triturated using fire-polished glass pipettes of decreasing bore sizes (600, 350 and 150 μm for LGd, and 300 and 150 μm for cortical samples). For triturating larger region of interest (ROI’s), pipets with three decreasing bore sizes were used (600, 300 and 150 µm). The cell suspension was incubated on ice in preparation for fluorescence-activated cell sorting (FACS).  **Note- Samples collected after 12/16/2016 had 0.0132M trehalose added to all solutions used after the point of slicing to improve cell viability and yield.
FACS preparation involved adding 2 μl of 4’-6-diamidino-2-phenylindole (DAPI) (2 mg/ml) to the 1.0 ml or 1 μl to the 500 μl ACSF.I-1% FBS cell suspension. The suspension was then filtered through a fine-mesh cell strainer (35 µm for samples collected before 2/22/2017, 70 µm for samples collected after 2/22/2017) and sorted by excluding DAPI positive events and debris, and gating to include red fluorescent events (tdTomato-positive cells). Single cells were collected into strip tubes containing 11.5μl of collection buffer (SMART-Seq v4 lysis buffer 0.83x, (Clontech #634894), RNase Inhibitor (0.17U/µl) and ERCCs (External RNA Controls Consortium) (Baker et al.; Risso et al.) (MIX1 at 1x10-8)). After sorting, the cells were subjected to centrifugation and then stored at -80°C.