We used ~10 whole adult DGRP379 and DGRP732 males and females. Flies were homogenized with an electric pestle in DNA/RNA Shield solution (Zymo Research). Homogenized tissue was digested with Proteinase K and RNA was purified with the Zymo Quick-RNA Plus Kit (Zymo Research). Ribosomal RNAs were removed using siTools rRNA depletion Kit (Galen Laboratory Supplies) and MyOne Streptavidin C1 Dynabeads (ThermoFisher) (#65001). Ribosomal RNA-depleted RNA was purified using the RNA Clean and Concentrator-5 kit (Zymo Research). Illumina libraries were generated using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB).
RNA sequencing reads were first aligned to the FlyBase r6.27 rRNA sequences using HISAT2 [77 (link)]. Non-ribosomal sequences were subsequently aligned to FlyBase r6.27 transcript sequences using htseq-ct [78 (link)]. Counts were filtered to include only expressed transcripts using DESeq2 [79 (link)] (rowSums(DESeqDataSetFromHTSeqCount) > = 1), which were subsequently normalized using rlog transformation (blind = TRUE) in DESeq2 [79 (link)].
RNA sequencing reads were first aligned to the FlyBase r6.27 rRNA sequences using HISAT2 [77 (link)]. Non-ribosomal sequences were subsequently aligned to FlyBase r6.27 transcript sequences using htseq-ct [78 (link)]. Counts were filtered to include only expressed transcripts using DESeq2 [79 (link)] (rowSums(DESeqDataSetFromHTSeqCount) > = 1), which were subsequently normalized using rlog transformation (blind = TRUE) in DESeq2 [79 (link)].