GSM6268033: QX2; Mus musculus; RNA-Seq
The cortex, midbrain and cerebellum were dissected and rapidly frozen in liquid nitrogen and stored at -80 degrees. Total RNA was extracted from the entire tissue from each animal using TRIZol Reagent (Life Technologies) and RNA clean and concentrator-5 with DNase I (Zymo Research) according to manufacturer's directions. mRNA was isolated from the total RNA using an NEBNext Poly(A) mRNA Magnetic Isolation Module as per instructions (New England Biolabs). cDNA libraries were generated from the isolated mRNA using an NEBNext Ultra RNA library prep kit for Illumina (New England Biolabs) and 8 nucleotide i7 and i5 dual-index primers (NEBNext multiplex oligos for Illumina, Dual Index Primers) were incorporated into each sample.  The library construction was done according to NEB instructions, modified for use with a Beckman Biomek 4000 at the Florida State University Biological Sciences core lab.  The unique dual-index (barcode) primers were added to each library to multiplex the forty-two libraries. The multiplexed sample was quantified with qPCR (Kapa Biosystems) specific for Illumina sequencing primers and the average fragment size was determined with a Bioanalyzer high sensitivity DNA chip (Agilent Technologies). The pooled sample was sequenced, with paired-end, 50 base reads on an Illumina NovaSeq 6000 located in the Translational Science Laboratory at the College of Medicine, Florida State University.  The pooled data were demultiplexed into individual sample data.