Mouse tissue harvest for snRNAseq.

Animal work was performed in accordance with protocols approved by MIT’s Committee on Animal Care and NIH guidelines. All postnatal mice were wild-type C57BL/6J originally obtained from Jackson Laboratories and bred in-house. Timed pregnant C57BL/6J females were either obtained from Jackson Laboratories to arrive between gestation day 11 and 15 or were impregnated in house by setting up overnight mating pairs with females in proestrus or estrus phase. Embryos were harvested at E18.5 (18 days after the plug date). Mice were housed in a facility with a light cycle running from 07:00 to 19:00, temperature 20-22.2°C, humidity 30-70%, and food and water available ad libitum. Postnatal mice were not derived from timed pregnant females. Instead, age was determined during regular pup checks by experienced researchers based on the Jax Mice Pup Appearance Chart. Thus, ages are approximate within +/− 0.5 days for P4 neonates, within +/−1 day for P14 early adolescents, within +/− 3 days for P32 juvenile mice and P90 young adult mice, and within +/−1 week for aged mice (90 weeks). Except for the P32 and aged time points, mice were obtained from different litters, and minimal replicate effects were observed in the snRNAseq data, suggesting adequate matching of developmental time points across replicates. A list of mice used in this study is provided in Table S1.

Non-neonate animals were acclimated to the lab space for at least 30 minutes prior to beginning euthanasia. Euthanasia took place between 9am-12pm to control for circadian rhythm effects, with a maximum of four animals processed per batch. Non-neonate animals were deeply anesthetized with isoflurane and decapitated. Heads were briefly submerged in liquid nitrogen for 3 seconds. Neonates were anesthetized via hypothermia and decapitated. Surgical tools were autoclaved and allowed to cool before use. All tools, brain mold, and the dissecting block were cleaned with RNase Zap^™ wipes prior to each dissection. Brains were harvested and sagittally sectioned at 1mm thickness for a total of 2 mm from the midline for either hemisphere (total of four ~1mm slices), on a brain mold using chilled razor blades. For P4 animals, two ~2mm sections from the midline were used. Tissue was dissected from slices on a chilled dissecting block exposed to room air, using a dissecting microscope at 1.6X magnification. Regions of interest were identified using the Allen Institute reference brain atlas at the appropriate time point. Dissected tissue was placed in cooled 1.5mL microcentrifuge tubes and spun down in a tabletop mini centrifuge prior to snap freezing in liquid nitrogen before storage at −80°C until nuclei isolation. 2-3 neonates were pooled in each tube. Time from decapitation to snap freezing ranged from 7-13 minutes per animal. Samples from at least 3 mice (at least 1 female) are represented at each developmental time point (except 90 weeks) and for each brain region. However, due to failures during microdissection, nuclei isolation, and 10x Genomics chip running, not all biological replicates are balanced across brain regions (e.g., some replicates have only one brain region present).

For embryonic brain microdissection, the pregnant dam was deeply anesthetized with an overdose of isoflurane, decapitated, and placed on a cooled dissecting block. The abdomen was opened and placentas were removed from the abdominal cavity. Embryos were harvested from the placenta and rapidly decapitated one-by-one. Heads were frozen on metal disks over dry ice for 5-10 minutes until frozen solid, stored at −20°C for 1.5 hours prior to microdissection. Heads were cut approximately in half using a small mouse brain mold and placed on a dry-ice cooled metal platform. Regions of interest were dissected using a tissue punch (1.27mm Ted Pella MilTex Biopsy Punch with Plunger, 15110-10) to extract tissue from most medial surface on either hemisphere. 2-3 embryos were pooled in each tube. The other dissection procedures were the same as described above. Dissections were performed in less than 20 minutes per set of tubes from decapitation to snap freezing.