title: GSM8775258: CeA Sample From Mouse 185; Mus musculus; RNA-Seq
library_construction_protocol: 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. Frozen tissue samples were transferred to the Gene Profiling Shared Resource at Oregon Health & Science University for total RNA isolation, using QIAsymphony RNA extraction methods for fatty tissue (QIAsymphony RNA Handbook, November 2020; Qiagen, Redwood City, CA, USA). Quality of the RNA was assessed by UV absorption (NanoDrop 8000) and all CeA samples had an average 260/280 ratio of 2.3, indicating high sample purity. RNA Integrity Number scores were obtained using a Bioanalyzer assay and ranged from 9.1 – 10.0, consistent with high quality RNA. Samples were transferred to the Massively Parallel Sequencing Shared Resource (MPSSR) for RNA-Seq data collection. The total number of samples was 200; 47-52 per lineper sex. The MPSSR completed library formation and sequencing according to Illumina's specifications. Stranded libraries (polyA+) were prepared using the TruSeq Stranded mRNA kit (Illumina, San Diego CA, USA) and were multiplexed in five batches of 40 on an Illumina NovaSeq 6000, yielding approximately 20 million paired-end reads per sample. Batches were balanced for sex and line.