library_construction_protocol: One hour after systemic injections of saline or morphine (10mg/kg) rats were euthanized by live decapitation and whole brains were briefly flash frozen in 2-methyl butane. Chilled brains were then blocked into 1-mm-thick coronal sections on a combination of dry and wet ice. Sections containing the VTA (ranging from -4.92-6.46 anterior-posterior (AP) to Bregma) were transferred to glass microscope slides on dry ice, and the VTA was microdissected away from neighboring brain regions (n = 3-4/sex/treatment group). Dissected VTA tissue was then transferred to ice-cold centrifuge tubes, and stored at -80°C until the day of 10X GEM capture. Frozen microdissected VTA tissue was prepared as previously described (Phillips et al., 2022). Briefly, tissue was thawed on wet ice and chopped with a scalpel prior to being transferred to 5 ml of ice cold lysis buffer (10 mM tris-HCl, 10 mM NaCl, 3 mM MgCl2 and 0.1% Igepal in nuclease-free water (Sigma-Aldrich, 18896-50ML). After 15 min the lysis was quenched with 5 ml of complete Hibernate A media (Thermo Fisher Scientific, A1247501) supplemented with B27, GlutaMAX (Life Technologies, 35050-061), and NxGen RNase Inhibitor (0.2 U/m; Lucigen, 30281-2). Tissue was then triturated by fire-polished Pasteur pipette (three pipettes of decreasing diameter) and filtered with a 40µm pre-wet cell strainer. Samples were pelleted and then washed in nuclei wash and resuspension buffer (phosphate-buffered saline (PBS), 1% bovine serum albumin (BSA), and NxGen RNase Inhibitor). The resulting pellet was resuspended in 800 ml of wash buffer and 7-aminoactinomycin D (7-AAD; Thermo Fisher Scientific, 00-6993-50) was added prior to fluorescence-activated cell sorting (FACS) on a FACSAria instrument (70 µm nozzle, BD Biosciences). Immediately after FACS, nuclei were washed a final time at 250 rcf in 10 ml of supplemented Hibernate A containing 1% BSA and RNase Inhibitor for 5 min at 4C. Nuclei were brought to a concentration of 1,000 nuclei/µL. A total of 8,000 nuclei pooled from two rats (4,000 male nuclei/4,000 female nuclei from the same treatment group) and were loaded into individual wells of the Chromium NextGem Single Cell Chip (10x Genomics, catalog no. 10000121). Libraries were constructed according to manufacturer's instructions for Chromium Next GEM single cell 3' library and gel bead kit (10x Genomics, v3.1 single index, catalog no. 10000121, version 3 chemistry)