title: RNA-seq of NEGR1 transgenic mice: adult brain
design: Whole mouse brains including the olfactory bulb, brainstem and cerebellum were snap frozen in dry ice and stored at -80C prior to processing for RNAseq analysis. Frozen brains were transferred from the -80C freezer into a Petri dish on dry ice and cut in half along the sagittal midline. One half of the brain was transferred into a polypropylene tube containing RNAlater-ICE solution (Cat. #AM7030, Thermo Fisher Scientific) precooled in the -80C freezer and incubated overnight at -20C. Total RNA was then isolated from this tissue using the RNeasy Mini kit (Cat. No. 74104, Qiagen). Brain tissues were weighed, transferred to the lysis buffer RLT (Qiagen) with freshly added DTT and immediately disrupted and homogenized using a glass Potter homogenizer. The volume of homogenate containing 30 mg of tissue used for homogenization was calculated as (lysis buffer volume x 30 mg) / brain tissue weight (in mg). This volume was used to isolate RNA following the manufacturers instructions. The RNA concentration and purity were measured by Epoch/Nanodrop spectrophotometer. The integrity of the RNA was evaluated on an Agilent TapeStation / Bioanalyzer. RNA samples with 260/280 and 260/230 ratios within 1.8 - 2.2 range, and RNA integrity numbers > 8.5 were prepared for sequencing. Poly(A)tailed RNA was subsequently prepared by the Ramaciotti Centre for Genomics at the University of New South Wales (UNSW) using the Illumina stranded mRNA prep ligation kit and used to create libraries for the deep sequencing analysis. The sequencing using the NovaSeq 6000 system (Illumina) was performed in the Ramaciotti Centre for Genomics at UNSW.