Title Multi-generational mRNA sequence data of pooled Drosophila melanogaster male and female flies from populations selected for long or short night sleep

Our objective was to determine whether gene expression in Drosophila melanogaster selectively bred for long or short night sleep duration changes detectably across generations. To meet this objective, we performed transcriptional profiling of ten pooled whole adult individuals from four selected populations and two control populations across 13 generations. We quantified differential expression among selection scheme (long sleep, short sleep, or unselected control), generation (generation 0; then generations 2-13), and sex for each gene. Overall design: We created four Drosophila melanogaster populations selected for long and short night sleep duration and two unselected control populations. The Sleep Advanced Intercross Population (SAIP) was the outbred starting population used, which originated from lines of the Drosophila Genetic Reference Panel (DGRP) (Serrano Negron et al., G3, 2018, PMID: 29991508). We selected flies for long and short night sleep for 13 generations under controlled environmental conditions. We seeded the fly culture bottles with 25 male and 25 female parents each generation. We reared the flies on standard Drosophila food at 25°C, 60% humidity, and a 12:12-hour light:dark cycle. Each generation, we collected 100 virgin males and 100 virgin females from each of the six population bottles and maintained the virgins at 20 individuals to a same-sex vial for four days. We then measured sleep for four days. After the sleep measurements, virgin flies were frozen for RNA extraction at the same circadian time (12:00 pm). For the starting generation (0) and generations 2-13, we randomly froze virgin male and virgin female flies from the sleep assays for RNA extraction. Ten males and ten females were frozen in two same-sex replicates from each of the six populations to generate 24 mRNA sequence profiles per generation. RNA was extracted using Qiazol (Qiagen, Hilden, Germany), followed by phenol-chloroform extraction, isopropanol precipitation, and DNase digestion (Qiagen, Hilden, Germany). To purify RNA, we used Qiagen RNeasy MinElute Cleanup kits (Qiagen, Hilden, Germany). We constructed Poly-A selected stranded mRNA libraries using the Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA). We added unique barcode adaptors to each sample. We conducted paired end sequencing using pooled libraries on an Illumina HiSeq2500 for a minimum of 38 million 126 base read pairs. Reads were mapped to FlyBase release 6 version 07 of the Drosophila melanogaster genome. Mapped reads were counted at the gene level.