GSM7885745
RNA isolation and purification were carried out by the Toxicology Core Facility at the University of Florida. For the temporal lobe, ventral midbrain, and cerebellum, RNA was isolated and prepared for extraction as previously described by Kondratov et. al. Total RNA for each sample was then extracted using a QIAGEN RNeasy® Lipid Tissue kit (Cat #74804) according to the manufacturer's instructions. Each sample's RNA Integrity Number (RIN) was determined using the Agilent 2100 Bioanalyzer to ensure RIN for each sample was greater than seven. Post-RNA extraction, a quality-controlled cDNA library was prepared and carried out by the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) in the NextGen DNA Sequencing (NGS) Core facility. Synthesis of cDNA libraries required 50 ng of RNA per sample, and cDNA synthesis reactions were performed using the NEBNext® Strand- switch cDNA synthesis reagents (Cat #E6421). Reactions were first optimized using the RNA samples with the lowest RIN. Amplification reactions were performed using NEBNext® High Fidelity 2X PCR Master Mix (Cat# M0541) at the following conditions: denaturation 98℃ for 45 sec, then cycling 98℃ for 10 sec, 62℃ for 15 sec, 72℃ for 3 min, final extension at 65℃ for 5 min. Each cDNA product underwent quality control using the Invitrogen Qubit® 3.0. Quality control was further ensured by testing every fourth sample using the Agilent 2200 Tape Station; at least 5 Tape Station runs were completed. Libraries were amplified for 14 rounds then sequenced on the Illumina NovaSeq6000. Overall, this generated 1,418,598,125- total reads with percent quality score greater than 30. These same optimized conditions were used to synthesize and amplify double-stranded cDNA for all samples.