Directional RNA-seq Library Preparation

Total RNA was quality checked using a Fragment Analyzer, depleted of rRNA and cDNA libraries prepared (UltraII Directional RNA Library Prep kit, NEB). Tibialis anterior and myotube libraries used 500 ng, whereas choroid plexus libraries used 200 ng of total RNA. Sequencing was performed on the NextSeq500 Illumina platform. Fastq files were inspected using Fastqc. Differential gene expression studies used DESeq2 (Love et al. 2014 (link)). For splicing analysis, reads were aligned to the mouse genome (mm10) using STAR(v2.6.0a) (Dobin et al. 2013 (link)) and splicing was quantified using rMATS(v4.0.2) (Shen et al. 2012 (link)). The rMATS output tables were filtered with the R package maser, with cutoff criteria of average reads ≥5, FDR <0.05, and minimum change in splicing of 10% (|ΔΨ| ≥0.1). RNA-seq data sets are deposited in GEO under accession no. GSE137494.