GSM5130024: VTA_Female_1_snRNA-seq; Rattus norvegicus; RNA-Seq
Rats were euthanized by decapitation, and brains were rapidly removed and chilled in ice cold Hibernate A media without Ca2+ or Mg2+ (BrainBits, HACAMG500) for 30-60 sec. Chilled brains were then blocked into 1-mm-thick coronal sections on wet ice. Sections containing the VTA (ranging from -4.92 to -6.46mm AP relative to bregma) were transferred to glass petri dishes on dry ice, and the VTA was microdissected from neighboring brain regions (n = 6 rats per sex).  VTA tissue was then transferred to ice-cold centrifuge tubes, and stored at −80°C until the day of nuclei isolation and harvesting. To harvest nuclei, frozen VTA tissue was briefly thawed on wet ice before being chopped by scalpel 100 times in two orthogonal directions. Tissue from three rats per sex was combined and 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) for 15 min, mixing the tissue by inversion every 2 min. Lysis was quenched after 15 min with 5 ml of complete Hibernate A (Thermo Fisher Scientific, A1247501) supplemented with B27, GlutaMAX (Life Technologies, 35050-061), and NxGen RNase Inhibitor (0.2 U/μl; Lucigen, 30281-2). Tissue was then triturated by fire-polished Pasteur pipette (three pipettes of decreasing diameter; 8 to 10 passes per pipette) for 35 min and passed through a 40-μm prewet filter. The samples were then pelleted at 500 rcf for 10 min at 4°C followed by a wash in 10 ml of nuclei wash and resuspension buffer [1× phosphate-buffered saline (PBS), 1% bovine serum albumin (BSA), and NxGen RNase Inhibitor]. Supernatant was then removed, and the pellet was gently resuspended in 800 μl of wash buffer before 7-aminoactinomycin D (Thermo Fisher Scientific, 00-6993-50) staining and fluorescence-activated cell sorting (FACS) to further purify the nuclei for sequencing (BD FACS Aria, 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 for 10 min at 4°C to remove any remaining fine debris and nascent RNA. Nuclei were then brought to a concentration of 1400 nuclei/μl. A total of 11,367 nuclei pooled from three rats per sex and treatment group were loaded into a single well of the Chromium Single Cell B Chip using four of the eight available wells. FACS-sorted nuclei were subject to 10X GEM generation and barcoding, GEM-RT cleanup and cDNA amplication, and library construction using a Chromium Single Cell 3ʹ Reagent Kit (v3 chemistry). Libraries were quantified and underwent sequencing (~500M total 75bp paired-end reads) on an Illumina sequencing platform (NextSeq 500).