GSM8887545: snATAC-Seq of CeA tissue from Mouse 12; Mus musculus; ATAC-seq
Brain tissue was obtained from experimentally naïve, non-ethanol exposed, S5 HP and LP mice. Mice were 60-70 days of age at the time of tissue harvesting (mean ± SEM = 63 ± 2 days). Mice group-housed by sex (2-4 per cage) were moved to a procedure room at 1000 h and allowed to acclimate for one hour; brains were obtained at 1100 - 1300 h. Mice were euthanized via cervical dislocation immediately followed by decapitation. Brains were removed using RNase-free tools, immediately frozen on dry ice, placed in RNase/DNase free polypropylene tubes and stored at -80°C until dissected. Frozen brains were dissected using anatomical landmarks from Franklin and Paxinos [85]. The CeA was obtained bilaterally, using RNase free tools, an aluminum dissection stage, and micropunches fashioned from blunt-cut 27 gauge needles. The frozen brain was placed ventral side up on the dissection stage and cut using a razor blade just anterior to the posterior boundary of the hypothalamus at bregma -0.82 mm. A second cut posterior to the first was then made at bregma -1.46 mm to obtain a 0.6 mm slice. The CeA was removed from this slice using the forked end of the external capsule, the lateral globus pallidus, and the internal capsule as landmarks. The bilateral CeA punches for each animal were placed in RNase and DNase free microcentrifuge tubes and stored at -80°C until RNA was extracted. snRNA-Seq libraries were generated from 16 S5 generation mice (n=4/line/sex). Flash-frozen CeA was dissociated into individual nuclei following manufacturer's instructions (protocol CG000366, 10X Genomics). Nuclei were filtered through a 40-70 mm mesh filter and centrifuged in a 29-50% OptiPrep gradient (Sigma-Aldrich) to separate intact nuclei from debris. Cells were inspected in a counting chamber for intact, bright, nongranular cell morphologies, indicating high viability and successful debris removal. ~ 10,000 single nuclei were captured for each sample in a single channel on the 10X Chromium controller, and snRNA/ATAC-Seq libraries (Chromium Next GEM Single Cell Multiome ATAC + Gene Expression) were generated following the manufacturer's instructions (10X Genomics). Libraries were sequenced to an average depth of 20,000-25,000 read pairs per nucleus (RNA-Seq and ATAC-Seq, respectively), in a NovaSeq6000 at the MPSSR according to 10X Genomics' specifications. All samples were processed and sequenced at the same time to avoid batch effects. Before filtering and clustering, we captured a total of 87,570 cells with a median of 4,890 UMI counts per cell and 2,320 genes per cell.