RNA extraction, RNA-seq and biological pathway analysis

Total poly-A-containing messenger RNA from hippocampi of 3 months Camk2a-Cre;tardbp^F/F and Camk2a-Cre;Tardbp^+/+ mice was immediately extracted using an RNeasy Mini kit (Qiagen, 74,104) hybrid protocol previously established in our lab [²⁷]. RNA was submitted to the Johns Hopkins Deep Sequencing & Microarray Core Facility for construction of 6 Illumina libraries. Transcriptome sequencing was performed using Illumina HiSeq2000. RNAseq analysis was performed using the Tophat suite on the Galaxy Project [⁵⁰–⁵²], a web-based bioinformatics platform. Output BAM files from Tophat were then used to calculate fold changes for different transcripts using the Cufflinks software suite [⁵³]. BAM files were also converted to BigBed tracks and mapped to the UCSC Genome Browser for graphical visualization. Analysis of the Camk2a-Cre;tardbp^F/F vs Camk2a-Cre;Tardbp^+/+ differentially expressed gene set was performed using the web-based Database for Annotation, Visualization and Integrated Discovery (DAVID, v.6.7). Statistically significant functional annotations were used to identify affected protein/gene pathways. The raw RNA-Seq file (SRX331737) was uploaded onto the Sequence Read Archive.