Differential gene expression analysis on forebrain,midbrain and hindbrain tissues dissected from 3 biological replicates of P-25 wild type and Fmr1CRE knockin mutant mice

RNA Sequencing analysis was performed on RNA isolated from tissues(forebrain,midbrain and hindbrain) from P-25 wild type and Fmr1CRE knockin mutant mice from same litter.Random primed cDNA from poly(A) selected RNA was converted into an Illumina sequencing library using RNA Library Prep Kit from Illumina (E7420, NEB, USA) in conjunction with NEBNext® Multiplex Oligos for Illumina (E7335/E7500, NEB, USA). and single-end 50-base pair (bp) reads were generated using a NextSeq 500 (Illumina Inc, SY-415-1002). Eighteen libraries were combined in two equimolar pools of 9 based on the library quantification results and each pool was run across a single High-Output Flow Cell. Sequencing was performed at the Wellcome Trust Clinical Research Facility (WTCRF; Edinburgh). Fastq files were processed to transcript-level counts and quality control performed using the bcbio_nextgen pipeline and the illumina-RNAseq workflow template. Differential Expression (DE) analysis was performed in R. The R package bcbio-RNAseq was used to import salmon transcript level counts into DESeq2. 18 samples , with 6 groups(2 genotype and 3 samples each genotype) and 3 replicates per group.