title: GSM6923106: Saline_F; Rattus norvegicus; RNA-Seq
library_construction_protocol: 1mm brain sections were taken at the level of the nucleus accumbens, and nucleus accumbens tissue was dissected using a 2mm punch tool centered on the nucleus accumbens core subregion. Frozen tissue punches were thawed slowly on ice prior to being chopped by scalpel 100 times in 2 orthogonal directions. Tissue from 4 animals in the same treatment condition was then combined and transferred to 5mL of chilled (4°C) Lysis Buffer (10mM Tris-HCl, 10mM NaCl, 3mM MgCl2 (Thermo Fisher, BP214-500), 0.1% Igepal in Nuclease-Free Water (Sigma, 18896-50ML)) for 15 min, mixing the tissue by inversion every 2 min. Lysis was quenched immediately at 15 min with 5mL of complete Hibernate-A (Thermo Fisher, A1247501) supplemented with B27 and Glutamax (Life Technologies, 35050-061)). Tissue was then triturated by fire-polished Pasteur pipette (2 pipettes of decreasing diameter; 8-10 passes per pipette) for 35 min and passed through a 40µm pre-wet filter. The samples were then pelleted at 500rcf for 10 min at 4°C followed by a wash in 10mL of Nuclei Wash & Resuspension Buffer (1X PBS, 1% BSA, 0.2U/ul NxGen RNase inhibitor (Lucigen, 30281-2)). Supernatant was then removed and the pellet was gently resuspended in a total volume of 800µl prior to 7-AAD (Thermo Fisher, 00-6993-50) staining and FACS to further purify the nuclei for sequencing. Immediately after FACS, nuclei were washed a final time at 200rcf in 10mL supplemented Hibernate-A containing 1% BSA for 10 minutes at 4°C to remove any remaining fine debris and nascent RNA. Nuclei were brought to a concentration of 1,500 nuclei/µl and an average total of 12,500 nuclei pooled from 4 rats were loaded per sex and treatment group. FACS-sorted nuclei were subject to 10X GEM generation and barcoding, GEM-RT cleanup and cDNA amplification, and library construction using a Chromium Single Cell 3ʹ Reagent Kit (v3 chemistry). Libraries were quantified and underwent sequencing (~550M total 75bp paired-end reads) on an Illumina sequencing platform (NextSeq 500).