title: GSM5467475: Grn-/-_3 hippomcampal nuclei; Mus musculus; RNA-Seq
library_construction_protocol: Nuclei were isolated with Nuclei EZ Prep Kit (SIGMA #NUC-101). Frozen hippocampus was homogenized using a glass dounce tissue grinder (SIGMA #D8938) 25 times with pastel A and 25 times with pastel B in 2 mL of ice-cold lysis buffer (Nuclei EZ lysis buffer with Complete Mini EDTA-free and Protector RNase Inhibitor (ROCHE)) and transferred to 15 mL tube with additional 2 mL of ice-cold lysis buffer, and then vortexed and incubated on ice for 5 min. Nuclei were centrifuged at 500 x g for 5 min at 4°C, washed with 4 mL of ice-cold lysis buffer, briefly vortexed and incubated on ice for 5 min. After centrifugation at 500 x g for 5 min at 4°C, the nuclei were washed with 4 mL of ice-cold nuclei suspension buffer (0.5 mg/mL BSA (ThermoFisher #AM2616) and 0.1% Protector RNase inhibitor in DPBS (CORNING #21-031-CV)) once, resuspended in 1 mL of nuclei suspension buffer, filtered through a 35 um cell strainer (FALCON #352235) twice, and counted with trypan blue. The samples were diluted to a final concentration of ~1,000 nuclei per uL and used for subsequent analyses. Nuclei of three samples ((WT, Grn+/−, and Grn−/−) or (PS19, PS19 Grn+/−, and PS19 Grn−/−)) were isolated and submitted for the sequencing at a time and the process was repeated six times to obtain three biological replicates for each genotype Libraries were prepared using Chromium Single Cell 3” Reagent Kits v3 (10X Genomics) according to the manufacturer's protocol.