GSM2197562: Control Rep3; Mus musculus; RNA-Seq
Mice were euthanized under anesthesia and the hypothalamus and medial amygdala were quickly dissected over ice. RNA was extracted following published protocols on translating ribosome affinity purification (TRAP). Isolated RNA was submitted to the Genomic and RNA Profiling Core at Baylor College of Medicine. Sample quality checks using the NanoDrop spectrophotometer and Agilent Bioanalyzer 2100 were conducted. Using NuGEN Ovation RNA-Seq System v2, purified double-stranded cDNA was generated from 10 ng of total RNA and amplified using both 3’ poly (A) selection and random priming. 2 µg of each sample was sheared using the Covaris S2 focused-ultrasonicator following the manufacturer’s protocol to obtain a final library with insert size of 400 bp. The sheared samples were quantified using the NanoDrop ND-1000 spectrophotometer and Invitrogen Qubit 2.0 DNA quantitation assay. The fragment sizes were confirmed on the Agilent Bioanalyzer to verify proper shearing. A double-stranded DNA library was produced using Illumina TruSeq DNA library preparation system and the sequencing was run on a HiSeq 2500 system.