Age- and ApoE genotype-dependent transcriptomic responses to O3 in the hippocampus of mice
Age- and ApoE genotype-dependent transcriptomic responses to O3 in the hippocampus of mice
Mice were euthanized by isoflurane overdose followed by bilateral thoracotomy. Brains were dissected sagittally into right and left hemispheres. The right hemisphere was fixed in 10% phosphate-buffered formalin, while the left hemisphere was dissected to isolate the hippocampus and cerebral cortex, which were snap-frozen in liquid nitrogen. mice were euthanized by isoflurane overdose followed by bilateral thoracotomy. Brains were dissected sagittally into right and left hemispheres.  Three-month-old (3M) and seventeen-month-old (17M) male human E4 and E3 gene targeted replacement (TR) mice were obtained from Taconic and housed at the University of Alabama at Birmingham (UAB).  Mice were subjected to either eight cycles of O₃ exposure or filtered air (FA) at the UAB environmental exposure facility. Each cycle consisted of 5 days of FA/O₃ exposure (0.8 ppm), for 7 hours per day followed by 9 days of FA. A total of 14 to 16 mice were used per genotype, age, and treatment group. Total RNA was extracted from the hippocampus using TRIzol reagent (Invitrogen; Ther-mo Fisher Scientific, Inc.) [59]. RNA concentration and purity were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and RNA integrity was evaluated using the RNA Nano 6000 Assay Kit on the Agilent Bi-oanalyzer 2100 system (Agilent Technologies, CA, USA). A total of 1 µg RNA per sample was used for library preparation using the NEB-Next® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) according to the manu-facturer's protocol. mRNA was purified using poly-T oligo-attached magnetic beads, fragmented under elevated temperature, and reverse transcribed into cDNA using random hexamer primers. The second-strand cDNA was synthesized using DNA polymerase I and RNase H, followed by end repair, adaptor ligation, and size selection (150–200 bp). Amplified libraries were purified and quality-checked using the Agilent Bioanalyzer 2100.