Bilateral VTA punches from two animals of the same sex and condition were pooled together and Dounce homogenized. Pairs were selected randomly from within each sex and condition. Pooling ensured a high enough concentration of nuclei per sample for quality sequencing. The sample was filtered through a 40um strainer and nuclei were isolated via iodixanol gradient centrifugation. Nuclei were hand-counted using a hemocytometer on an EVOS M5000 microscope and final concentrations were 850-1,450 nuclei per ul. The volume of suspension used for library preparation was adjusted in order to load a target of 20,000 nuclei and capture 10,000 nuclei per sample. Library construction was performed according to the manufacturer's instructions (10X Genomics Chromium v3). Briefly, nuclei were resuspended in the master mix and loaded together with partioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the lysate contained in every single GEM was retrotranscripted to cDNA, which contains an illumina R1 primer sequence, unique molecular identifier (UMI), and the 10X barcode. The pooled and barcoded cDNA was then cleaned up with Silance DynaBeads, amplified by PCR, and the appropriate sized fragments were selected qith SPRIselect reagent for subsequent library construction. During library construction the Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences, and a sample index were added.