title: GSM6470105: Central amygdala biological replicate 2; Mus musculus; RNA-Seq
library_construction_protocol: All sacrifices were performed during the dark period of the light cycle. Animals were anesthetized via combined intraperitoneal injection of 150 mg/kg ketamine and 15 mg/kg xylazine. Once unconscious, mice animals were transcardially perfused with ice-cold, carbogen-bubbled (95% O2, 5% CO2), nuclease-free, 0.22 µm sterile-filtered artificial cerebrospinal fluid (ACSF) with a composition of 93 mM N-methyl-D-glucamine, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 5 mM sodium ascorbate, 2 mM thiourea, 3 mM sodium pyruvate, 13.2 mM trehalose, 12 mM N-acetyl-cysteine, 0.5 mM CaCl2, 10 mM MgSO4, and 93 mM HCl, at pH 7.3-7.4.(Tasic et al., 2018; Ting et al., 2014) Following transcardial perfusion, brains were immediately extracted and submerged into ice-cold carbogen-bubbled ACSF, with less than 5 minutes between the beginning of perfusion and final submersion after extraction. Brains were serially sectioned in ice-cold, carbogen-bubbled ACSF on a VT1000S vibratome (Leica) with polytetrafluoroethane-coated razor blades (Ted Pella) at 0.15 mm/sec and 100 Hz, dividing the whole cerebrum into 400 µm coronal slices. Target regions were microdissected from these slices under a stereomicroscope using a sterile blunt-end needle (22 gauge for CeA, ASt, and tail of striatum, 16 gauge for dorsal striatum). All regions were targeted using Paxinos and Franklin's the Mouse Brain in Stereotaxic Coordinates as reference (Paxinos and Franklin, 2019). Extracted tissue samples were recovered in ice-cold, nuclease-free, 0.22 µm sterile-filtered cryoprotective nuclear storage buffer, composed of 0.32 M sucrose, 5 mM CaCl2, 3 mM magnesium acetate, 10 mM Trizma hydrochloride buffer (pH 8.0), 1 mM dithiothreitol, 0.02 U/µl SUPERase•In RNAse Inhibitor (Invitrogen), and 1X cOmplete Protease Inhibitor Cocktail with EDTA (Roche). Tissue was then snap frozen using a metal CoolRack M90 (Biocision) pre-chilled to -80˚C and stored at -80˚C until nuclear isolation. Cryopreserved tissue pieces were slow-thawed by incubation at 4˚C for 1 hour prior to isolation. Tissue pieces were then pooled and resuspended in nuclear isolation medium composed of 0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Trizma hydrochloride buffer (pH 7.4), 1 mM dithiothreitol, 0.04 U/µl RNasin Plus RNAse Inhibitor (Promega), 1X cOmplete Protease Inhibitor Cocktail with EDTA (Roche), and 0.1% Triton-X. The pooled tissue pieces in nuclear isolation medium were transferred to a 2 mL Dounce tissue grinder. Tissue was homogenized by 5 strokes from the loose pestle and 15 followed by the tight pestle, and the resulting homogenate was filtered through a 40 µm Flowmi cell strainer (Bel-Art) into a 1.5 ml Lo-Bind tube (Eppendorf). The homogenate was then centrifuged with a swinging bucket rotor at 4˚C and 1000 x g for 8 minutes. Nuclei were then washed with nuclear flow buffer composed of DPBS with 1% bovine serum albumin, 1 mM dithiothreitol, and 0.04 U/µl RNasin Plus RNAse Inhibitor (Promega) and centrifuged at 4˚C and 500 x g for 5 minutes, which was subsequently repeated. Nuclei were finally resuspended in nuclear flow buffer containing 3 µm DRAQ7 (Cell Signaling Technology) and again filtered through a 40 µm Flowmi cell strainer into a 5 ml round-bottom polystyrene tube. Each isolation took under 45 minutes to perform, from homogenization to final suspension. Library construction was performed acording to manufacturers' instructions (10X Single Cell 3' v3.1 Single Index). GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip. RNA from nuclei in each droplet was reverse-transcribed to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with DynaBeads, PCR-amplified and appropriately-sized fragments were selected with SPRIselect reagents for subsequent library construction. During subsequent steps, Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.