GSM8795031: Arc-negative NAc nuclei, CPP 5 pairing, homecage; Mus musculus; RNA-Seq
Mouse brains were collected after cervical dislocation and bilateral NAc dissection was rapidly performed from 1 mm-thick coronal brain sections using a 14G needle and tissue frozen on dry ice. Bilateral dissections from 16-20 mice were pooled together group-wise into one single sample, which did not allow for the use of pseudobulking analysis strategies (see below). Nuclei suspension was obtained by homogenization of frozen pooled NAc samples in 4 mL of low-sucrose lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM Mg(Ace)2, 0.1 mM EDTA, 10 mM Tris-HCl) using a large clearance then a small clearance pestle of a glass Dounce tissue grinder (Kimble Kontes). The homogenates were filtered in an ultracentrifuge tubes (Beckman Coulter through a 40 μm cell strainer (Pluriselect) into ultracentrifuge tubes (Beckman Coulter), underlaid with 5 mL of high sucrose solution (1.8 M sucrose, 3 mM Mg(Ace)2, 1 mM DTT, 10 mM Tris-HCl). After centrifugation at 107,000 g for 1 h at 4°C in a SW41Ti Swinging-Bucket Rotor (Beckman Coulter, the supernatant was discarded, and nuclei pellets were resuspended in 800 µL of a solution containing 0.5% bovine serum albumin (BSA) in PBS and supplemented with DAPI at 1 µg/mL to allow for nuclei detection. Nuclei were sorted on a BD FACS Aria II three-laser device with a 100 μm nozzle and using BD FACSDiva Software v8.0.2. Gating strategies from a representative sort are visualized in Extended Data Fig. 7. Briefly, debris and doublets were excluded using FSC and SSC filters, nuclei were then selected as DAPI-positive (Violet1-A laser) events, and finally GFP-positive 610 nuclei (Blue1-A laser) were sorted directly into BSA-coated low-binding tubes. 15-18,000 nuclei were recovered for GFP+ samples and 100,000 for GFP- samples. Following FANS, nuclei were quantified (Countess II, Life Technologies) and ±5,000 per GFP+ sample and ±20,000 per GFP- sample were loaded on a single 10X flow cell GEM well using Chromium Single Cell 3' Library Construction Kit (10X Genomics). cDNA libraries were prepared according to the manufacturer's protocol (10X Genomics). Libraries were sequenced at Azenta on a single NovaSeq lane (Illumina) at a depth of ±350 million reads per sample.