A holistic systems approach to characterize the impact of pre- and post-natal oxycodone exposure on neurodevelopment and behavior

RNA-sequencing of total RNA isolated from P14 PFC tissues of animals exposed to oxycodone or saline revealed alterations in the expression of key genes associated with synaptic transmission, neurodevelopment, mood disorders, and addiction in the treatment groups. Overall design: Total RNA from prefrontal cortex (PFC) tissue was isolated from the randomly selected pups (n = 6) from each treatment group at P14 using the Direct-Zol RNA kit (Zymo Research, CA, USA) based on the manufacturer's protocol. Samples were sent to UNMC's Next Generztion Sequencing (NGS) core. RNAseq libraries were generated beginning with 1 ug of total RNA from each sample using the TruSeq V2 RNA sequencing library kit from Illumina following recommended procedures (Illumina Inc., San Diego, CA). Resultant libraries were assessed for size of insert by analysis of an aliquot of each library on a BioAnalyzer instrument (Agilent Technologies, Santa Clara, CA). Each library contained a unique indexing identifier barcode allowing the individual libraries to be multiplexed together for efficient sequencing. Multiplexed libraries (18 samples per pool) were sequenced on a single flow cell of the NextSeq550 DNA Analyzer (Illumina) to generate a total of approximately 28 million 75 bp single reads for each sample.