Fresh dorsal hippocampal tissue was collected from mice 96 h after contextual fear conditioning (CFC). RNase inhibitor was added to the 6x Homogenization Buffer Stable Master Mix (30 mM CaCl2, 18 mM Mg(Ac)2, 60 mM Tris pH 7.8, in H2O) at a final concentration of 1.2U/μl. Tissue was homogenized using a Polytron homogenizer for 1.5 minutes. Samples were resuspended with the 50% iodixanol solution (50% iodixanol in 1× homogenization buffer) via gentle pipetting to make a final concentration of 25% iodixanol. 3ml of a 35% iodixanol solution (35% iodixanol in 1× homogenization containing 480 mM sucrose) was added to a new 15 ml Falcon tube. 3ml of a 29% iodixanol solution (29% iodixanol in 1× homogenization buffer containing 480 mM sucrose) was layered above the 35% iodixanol mixture. A 4ml 25% iodixanol solution was layered on the 29% solution. In a swinging-bucket centrifuge, nuclei were centrifuged for 20 min at 3,000 r.c.f. After centrifugation, the nuclei were present at the interface of the 29% and 35% iodixanol solutions. This band with the nuclei was collected in a 300 μl volume and transferred to a pre-chilled tube. Following isolation, nuclei were counted and resuspend in FACs buffer (1% BSA, 1mM EDTA, and 0.2U/μl RNase inhibitor in PBS) at 2 million nuclei per milliliter. A subset of nuclei was sorted by GFP expression on a high-speed cell sorter flow cytometer (Beckman Coulter MoFlowXDP cell sorter). snRNA-seq libraries were generated from up to 10,000 individual cells captured in an oil emulsion on a Chromium Controller (10x Genomics, Pleasanton, CA.) cDNA wasgenerated in the individual cell-gel bead emulsion micro-reactors while adding barcodes at the cellular and molecular level using the Chromium Next GEM Single Cell 3' Kit v3.1(10xGenomics kit 1000268.) The barcoded cDNAs from the individual cells were combined for the remaining library process. The unique molecular barcodes (UMIs) prevented amplification artifacts to skew the analysis. The libraries were analyzed on a Fragment Analyzer 5200 (Agilent Technologies, Santa Clara, CA) to ensure a normal sizedistribution with an average size of 450 bp and sequenced on an llumina Sequencer with the following read lengths: 28 bp for Read 1, 10bp for i7 Index,10bp for i5 Index and 90 bp forRead 2 at a read depth of 20,000 reads per cell.