GSM7882333: MN3NonEns; Mus musculus; RNA-Seq
Whole brains were extracted from euthanized mice (5% isoflurane exposure followed by cervical dislocation) and incubated for 5 minutes on ice in cellular stabilizing solution: 45% HEBG medium (2% 50X B27 (Thermo Fisher; 17504044) and 0.25% 100X Glutamax (Thermo Fisher; 35050061) per 1 mL of Hibernate E-Minus Phenol Red (Thermo Fisher; NC1506837)), 45% artificial cerebral spinal fluid (aCSF;  7.13 g/L NaCl (Sigma-Aldrich; S9888-500G), 0.23 g/L KCl (Sigma-Aldrich; P3911-25G), 0.14 g/L MgSO4 (Sigma-Aldrich; 746452-500G), 0.19 g/L CaCl2 (Sigma-Aldrich; C33067-100G), 0.048 g/L NaH2PO4 (Millipore-Sigma; 10049-21-5), 2.1 g/L NaHCO3 (Millipore-Sigma; 144-55-8), 1 L diH2O adjusted for dry reagent volumes), 9.97% RNAlater (Thermo Fisher; AM7020), and 0.03% RNaseOUT (Thermo Fisher; 10777019). Brain sections (1.94-0.64 bregma; 1.5 mm thickness) containing the nucleus accumbens core and prefrontal cortex were cut using a brain matrix (0.5 mm spaced slits) kept cold at -20°C with ice-cold razors. Punches of brain tissue were made using a 1 mm core puncher for each nucleus accumbens bilateral region and 1.5 mm sized punches for the prefrontal cortex. Brain regions from three mice per sex were pooled together for adequate RNA collection. Brain punches were immediately placed in enzymatic dissociation solution (2mg/mL papain (Worthington; LS003120) diluted in 1 mL HEBG with 0.125% 100x Glutamax – heated in 30C water bath for 10 minutes before immediately placing on ice for brain punches) on ice for 20 minutes. Mechanical trituration while incubating in papain solution was carried out using 10x syringe fluctuations with a 23G needle followed by a 27G needle. Caution is taken to not introduce air bubbles during syringe fluctuations that otherwise rupture cell membranes. Tissue was centrifuged for 3 minutes at 1000 rcf at 4°C, and the supernatant was carefully discarded via pipette, leaving ~20 uL medium to prevent significant cell loss. Cells were slowly resuspended via pipette with 500 uL of 4°C HEBG medium and then filtered through a 40 um cell strainer. Semi-permeabilization was carried out by adding 500 uL of -20°C ethyl acetate (95% ethanol; 5% acetate) to the 500 uL of HEBG medium with cells for a final 50% ethyl acetate concentration. Tubes were slowly inverted by hand for 1 minute briefly before centrifuging for 5 min at 1000 rcf in 4°C. The supernatant was discarded carefully via pipette leaving ~20 uL to prevent cellular loss and cells are resuspended gently via pipette in 500 uL of 4°C HEBG medium with neuronal (NeuN-Alexafluor-405; conc. 5:500; Novus Biologicals; NBP1-92693AF405) and cFos-labeling conjugated antibodies (cFos-Alexafluor-488; conc. 5:500; Novus Biologicals; NBP2-50037AF488). Cells were incubated at 4°C while rotating in the dark for 45 minutes. Cells were centrifuged for 5 minutes at 1000 rcf at 4°C, and the supernatant was carefully discarded via pipette, leaving ~20 uL medium to prevent significant cell loss. Cells were resuspended in 1 mL of 4°C HEBG medium and filtered with a 40 um cell strainer into a sterile 5 mL FACS tube and kept at 4°C. Cells were then sorted for neuronal and reward-specific tags (neuron – immunolabeled NeuN; cocaine – endogenous tdTomato; sucrose – immunolabeled cFos; overlap – tdTomato/cFos) using FACS (FACSMelody; FACSChorus Software 100-um nozzle; BD Biosciences). 80k-100k events per reward-specific population were sorted directly into the RLT buffer (RNeasy Mini Kit; Qiagen; 74104) for downstream RNA extraction using RNeasy Mini Kit. RNA samples were kept at -80°C until downstream RNA sequencing. RNA samples underwent library preparation and next-generation RNA sequencing at the University of Colorado Anschutz Medical Campus Genomics and Microarray Core. Low-input ribosome depleted (SMART-seq; Takara Bio; library size: 200-500bp) library prep was used for downstream paired-end 2x150bp RNA sequencing at a 50 million read depth using NovaSEQ 6000 (Illumina).