RNA-Seq Analysis of Mouse Brain Tissue

Brain tissue was homogenized using a GentleMACS dissociator and mRNA was isolated using the RNeasy mini kit (Qiagen GmbH, Hilden, Germany). RNA concentration (ratio 260/230 and 260/280 nm) was measured using a NanoDrop ND-1000 (Thermo Scientific, Waltham, MA, USA) and RNA integrity evaluated using an RNA screen tape on a Tapestation 2200 (Agilent, Santa Clara, California, USA). A bulk variation of MARSseq [16 (link)] was used to construct RNAseq libraries. Sequencing was performed using an Illumina Nextseq-500 75 cycle high output kit (Illumina, San Diego, California, USA; paired end sequencing). Raw reads were mapped to the Mus musculus genome (mm10) using STAR. Only reads which mapped uniquely to genes were considered for further analysis. Differentially-expressed genes (DEGs) were selected using a 1.5-fold change cutoff between two populations and adjusted p value for multiple gene testing of < 0.05 [17 (link)]. Pathway analysis was performed using Gene Analytics [14 (link)]. Ortholog conversion was done using HUGO Gene Nomenclature Committee.