Our primary objective was to characterize the amount of variation in transcript abundance among individual flies with identical genotypes. We also wanted to determine which analysis methods would be optimal for RNA-Seq data. To meet these objectives, we performed transcriptional profiling of whole adult individuals from 16 Drosophila Genetic Reference Panel (DGRP) lines. We quantified differential expression among genotypes, environments, and sexes.
Overall design: We randomly chose 16 DGRP lines for this experiment: DGRP-93, DGRP-229, DGRP-320, DGRP-352, DGRP-370, DGRP-563, DGRP-630, DGRP-703, DGRP-761, DGRP-787, DGRP-790, DGRP-804, DGRP-812, DGRP-822, DGRP-850, and DGRP-900. We collected 8 virgin male and 8 virgin female flies from the 16 DGRP genotypes in three replicated environments to produce RNA sequence profiles. We controlled the environmental conditions in the following ways. We seeded the fly cultures with 5 male and 5 female parents. We reared the progeny in a single incubator on standard Drosophila food (http://flystocks.bio.indiana.edu/Fly_Work/media-recipes/bloomfood.htm) at 25°C, 60% humidity, and a 12:12-hour light:dark cycle. We collected and maintained male and female virgins at 20 flies to a same-sex vial for four days prior to RNA extraction to control for social exposure. Flies were frozen for RNA extraction at the same circadian time (1:00 pm) in 96-well plates. PolyA RNA stranded libraries were prepared by modifying an existing protocol. ERCC (External RNA Controls Consortium, SRM2374, beta version, pools 78A/78B) sequences were added during the library preparation as a control. For some samples >1 library was generated to check technical variation. We performed multiplexed single-end 76 bp sequencing on an Illumina HiSeq2000. Reads were mapped to FlyBase release 5 version 57 and release 6 version 01 of the Drosophila melanogaster genome and the ERCC sequences. Mapped reads were counted at the gene level.
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