RNA libraries were polyA-enriched to identify mRNA transcripts ready for nuclear export and sequenced using a Hi-Seq 4000 (Illumina) machine at a depth of ∼30 million single-end 1 × 50 bp reads by GENEWIZ (South Plainfield, NJ). Read quality was assessed using FastQC 0.11.5 (Babraham Bioinformatics). Adapters were removed and trimmed using Trimmomatic 0.36 (Bolger et al. 2014). The “new tuxedo suite” was used to further process reads (Pertea et al. 2016). Reads were aligned with HISAT2 2.0.5 (Pertea et al. 2016) to the Ensembl BDGP6_transcriptome reference (Dm6), modified to include the 5xUAS-unc-84-2xGFP sequence present in our flies. Next, samtools 1.3.1 (Li et al. 2009) and Stringtie 1.3.3 (Pertea et al. 2016) were used to sort, merge, and quantify transcripts. Lastly, assembly and analysis of Ballgown objects (Pertea et al. 2016) and data visualization with ggplot2 was performed in RStudio 1.0.136 (Wickham 2009). No samples were discarded and default settings were used for the pipeline unless otherwise noted; command line code as follows:#Trimmomatic:TruSeq3-SE.fa:2:30:10:8:true LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36#HISAT2:hisat2 -q -x <reference index> -U <input fastq file> -S <output sam file> -p
