title: GSM5106144: 03_E3_10_H10; Mus musculus; RNA-Seq
library_construction_protocol: To isolate single nuclei from adult mouse brains, we combined and adapted the 10x Genomics demonstrated protocol for nuclei isolation from adult mouse brain55 and the Allen Brain Institute protocol for FACS sorting of single nuclei56 as follows. Hippocampi were acutely dissected on ice. Dissected hippocampi were placed in 2mL Hibernate A®/B27®/GlutaMAX™ (HEB) medium in a 5mL tube. The HEB medium was removed to a 15mL conical and kept on ice. 2mL of chilled lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, and 0.1% Nonidet™ P40 Substitute in Nuclease-Free Water) were added to the tissue, and the hippocampi were homogenized by suctioning 10 times through a 21G needle. After homogenization, the tissue was lysed on ice for 15min, swirling 2–3 times during this incubation period. The reserved chilled HEB media was then returned to the lysed tissue solution, and the tissue was further triturated with 5–7 passes through a 1mL pipette. A 30µm cell strainer (MACS SmartStrainer; Miltenyi Biotech 130-110-915) was washed with 1mL of PBS, and the lysed tissue solution was filtered through the strainer to remove debris and clumps. Filtered nuclei were centrifuged at 500 rcf for 5min at 4°C. The supernatant was removed, and nuclei were washed in 1mL of Nuclei Wash and Resuspension Buffer (1X PBS with 1.0% BSA and 0.2U/µl RNase Inhibitor). Nuclei were again centrifuged at 500 rcf for 5min at 4°C and resuspended in 400µL of Nuclei Wash and Resuspension Buffer. DAPI was added to a final concentration of 0.1ug/mL, and the nuclei were filtered through a 35µm cell strainer. DAPI-positive nuclei were sorted by gating on DAPI-positive events, excluding debris and doublets, using the BD FACSAria-II at the Gladstone Institutes' Flow Cytometry Core. cDNA libraries were prepared using the Chromium Single Cell 3′ Library and Gel Bead kit v2 (10x Genomics: 120267) according to the manufacturer's instructions. Libraries were sequenced on an Illumina NovaSeq 6000 sequencer at the UCSF CAT Core.