title: snRNA-seq of mouse: KO 16 days
design: Brains were collected from P16 mice and the forebrain was isolated and flash frozen in liquid nitrogen. Nuclei were isolated using a protocol adapted from Habib et al. (89). Forebrains were homogenized using a glass Dounce tissue grinder (25X with pestle A and 25X with pestle B) in 2 mL of EZ PREP buffer (Sigma NUC-101). Homogenized tissues were incubated on ice for 5 minutes with an additional 2 mL of EZ PREP. Nuclei were pelleted by centrifuging at 500g for 5 minutes at 4C. Nuclei were washed with 4 mL EZ PREP and incubated on ice for an additional 5 minutes. Nuclei were centrifuged again and washed in 4 mL of nuclear suspension buffer (0.01% BSA, RNase inhibitor [Takara 2313A]) followed by centrifugation for 5 minutes at 500g. The nuclear pellet was resuspended in 1 mL of nuclear suspension buffer before filtering through a 35 M cell strainer (Corning 352235) and counted. Nuclei were diluted to a final concentration of 800-1500 cell/L. Single-nuclear libraries were generated using the 10X Chromium Controller system. Libraries were sequenced using an Illumina NovaSeq 6000.