title: GSM8316436: lacZ-S8; Rattus norvegicus; RNA-Seq
library_construction_protocol: Brains were blocked in 1mm sections, and NAc punches were obtained, flash frozen, and stored at -80°C prior to nuclei isolation. 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, and frozen for storage. Frozen tissue punches were thawed slowly on ice prior to being chopped by scalpel 100 times in 2 orthogonal directions. Tissue from one rat was 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. 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) on the Chromium iX instrument. Libraries were constructed according to manufacturer's instructions using the Chromium Single Cell 3′ Library Construction Kit (10× Genomics, PN-1000190). Nuclei (6,221 total) from adult rat NAc were sequenced on the Illumina NovaSeq6000 at the UAB Heflin Genomics core to a depth of ~300,000 reads per nuclei.