For JBrowse display of FlyAtlas2 RNA-seq and microRNA RNA-Seq genomic coverage data, FlyBase started with bam files provided by the FlyAtlas2 group (reads from 2 biological replicates were aligned to the Release 6 reference genome, using the Tuxedo pipeline, and the resulting bam files were merged). FlyBase then used the bedtools genomecov function to generate bedGraph coverage data from bam files, then an in house FlyBase script to convert bedGraph coverage data to wig format. FlyBase FlyAtlas2 wig files are available here: https://ftp.flybase.net/flybase/associated_files/RNA-seq/FlyAtlas2_RNA-Seq/.

Reads were pseudo-aligned to all non-small RNA Drosophila melanogaster transcripts (r6.54) using SALMON (v1.10.1). A decoy transcriptome was created using the Drosophila chromosome sequences. Transcript-level abundances were summarized to the gene level using tximport (v3.18). Ribosomal (rRNA) transcripts, genes, and all aligned reads were removed from the datasets, to prevent biasing of FPKM values based on ribosomal content, which we found varied a great deal between samples due to batch effects. FPKMs were calculated using the standard calculation: FPKM = [RMg * 109 ] / [RMt * L], where RMg = The number of reads mapped to the gene, RMt = The total number of mapped reads (excluding removed genes), and L = The length of the gene in base pairs.