Ezna isolation kit

Pigs were anesthetized intramuscularly using a drug cocktail of telazol:ketamine:xylazine (50 mg of tiletamine; 50 mg of zolazepam) reconstituted with 2.5 mL ketamine (100 g/L) and 2.5 mL xylazine (100 g/L; Fort Dodge Animal Health, Fort Dodge, IA, USA) at a dose of 0.03 mL/kg body weight, following protocols (Antonson et al. 2017 (link)) at 22 days of age. An intracardiac injection of sodium pentobarbital (86 mg/kg body weight, Fata Plus, Vortech Pharmaceuticals, Dearborn, MI, USA) was used to euthanize the pigs after anesthetization. After euthanasia, the pig brains were removed, and the stereotaxic atlas of the pig brain (Félix et al. 1999 (link)) was used to identify the amygdalae. Amygdalae were dissected out, flash frozen on dry ice, and stored at −80°C following published protocols (Antonson et al. 2019 (link)). EZNA isolation kit (Omega Biotek, Norcross, GA, USA) was used to isolate RNA per manufacturer’s instructions. The RNA integrity numbers of the samples were above 7.3, indicating low RNA degradation. TruSeq Stranded mRNAseq Sample Prep kit’ (Illumina Inc, San Diego, CA, USA) was used to prepare RNAseq libraries, and libraries were quantitated by qPCR and sequenced on one lane on a NovaSeq 6000 for 151 cycles from each end of the fragments using NovaSeq S4 reagent kit. The bcl2fastq v2.20 conversion software was used to produce and demultiplex FASTQ files.

Pigs were anesthetized intramuscularly using a drug cocktail of telazol:ketamine:xylazine (50 mg of tiletamine; 50 mg of zolazepam) reconstituted with 2.5 mL ketamine (100 g/L) and 2.5 mL xylazine (100 g/L; Fort Dodge Animal Health, Fort Dodge, IA, USA) at a dose of 0.03 mL/kg body weight, following protocols [19 (link)] at 22 days of age. An intracardiac injection of sodium pentobarbital (86 mg/kg body weight, Fatal Plus, Vortech Pharmaceuticals, Dearborn, MI, USA) was used to euthanize pigs. The brains were removed, dissected, flash frozen on dry ice, and stored at −80 °C following published protocols [22 (link)]. From the hippocampi, an EZNA isolation kit (Omega Biotek, Norcross, GA, USA) was used to isolate RNA per the manufacturer’s instructions. An RNA integrity number of >7.3 was used as a cutoff for the samples to ensure low degradation. A TruSeq Stranded mRNAseq Sample Prep kit (Illumina Inc., San Diego, CA, USA) was used to prepare RNAseq libraries, and libraries were quantitated by qPCR and sequenced on one lane on a NovaSeq 6000 for 151 cycles from each end of the fragments using NovaSeq S4 reagent kit. The bcl2fastq v2.20 conversion software was used to produce and demultiplex FASTQ files.