Thermo scientific nanodrop lite spectrophotometer

For each animal, all-layer samples of thoracic and abdominal aortic tissue specimens were placed in RNAlater^® solution (Ambion, Austin, TX, USA) and stored at 4 °C for subsequent RNA extraction. Given that the atherosclerotic lesions were diffusely spread throughout the aortas, samples were randomly selected. In total, 100–200 mg of tissue was utilized from each experimental animal (50–100 mg thoracic aorta and 50–100 mg abdominal aorta). Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions [27 (link)]. RNA was quantified using a Thermo Scientific NanoDrop™ Lite Spectrophotometer (Thermo Scientific, Waltham, MA, USA). RNA quality was assessed by the A₂₆₀/A₂₈₀ ratio. Complementary DNA (cDNA) was produced when the A₂₆₀/A₂₈₀ ratio was greater than 1.8 [27 (link)]. Complementary DNA was synthesized using the PrimeScript RT reagent kit (Takara Bio, Kusatsu, Shiga, Japan), according to the manufacturer’s protocol.

DNA was further purified using the Bio-RAD Micro Bio-Spin Columns P-30 Tris following the manufacturer’s instructions. This results in 75 µl of purified DNA, which can be used for PCR. We then ran PCRs using the Qiagen multiplex kit (with 1.5 µl of molecular grade water), 5 µl of Qiagen multiplex PCR Master Mix, then 0.5 µl of 10 µM for the forward and reverse primers, and with the addition of 0.5 µl of bovine serum albumin (BSA) to counteract remaining PCR inhibitors and 2 µl of template DNA. The PCRs started with an initial activation period of 95 °C for 15 min, followed by 35 cycles with the following settings: denaturation at 94 °C for 30 s, the annealing temperature of 65 °C for 90 s; extension at 72 °C for one minute. A final elongation step was performed at 72 °C for 10 min. PCRs were repeated up to three times per sample. For all 177 beetles, we used vertebrate-specific PCR primers to amplify portions of the mitochondrially encoded 12S rDNA (Ushio et al., 2017 (link)). For the Ecuadorian beetles (N = 135 beetles), we additionally used the mammal-specific primers 16smama1 (forward) and 16smama2 (reverse) (Taylor, 1996 (link)). For a subset of samples (N = 121 DNA extractions), we measured the DNA concentration using a ThermoScientific Nanodrop Lite Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Of 177 beetles, 128 were amplified and sent to sequencing.

Following dropout from the ABA paradigm, mice were sacrificed via decapitation for rapid brain extraction. Using a brain matrix and a tissue punch, the mPFC (AP 2.56 to 2.06; ML ±0.25; DV -2.00 from bregma), HPC (AP -1.56 to -2.56; ML ±1.5; DV -2.00 from bregma), NAc (AP 2.06 to 1.06; ML ±0.50; DV -5.00 from bregma), and VTA (AP -3.06 to -4.06; ML ±0.50; DV -4.50 from bregma) regions were rapidly dissected and snap frozen on dry ice. Samples were stored at -80C prior to RNA extraction. Tissue was homogenized in Qiazol lysing reagent (Qiagen, Hilden, Germany) using Lysing Matrix D tubes (MP Biomedicals, Solon, OH, USA) and the MP Biomedicals FastPrep-24 tissue ruptor (MP Biomedicals, Solon, OH, USA). RNA was extracted using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany). Following RNA extraction, samples were analyzed for purity and RNA concentration using the Thermo Scientific NanoDrop LITE Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Samples were then diluted to achieve identical RNA concentrations for reverse-transcription PCR. Reverse-transcription PCR was performed using the Taqman Reverse Transcription kit (Life Technologies, Carlsbad, CA, USA). PCR cycle is as follows: 25°C 10min, 37°C 60min, 95°C 5min.