Identification of genes contributing to a long circadian period in Drosophila melanogaster. Identification of genes contributing to a long circadian period in Drosophila melanogaster

Our objective was to identify candidate genes that contribute to the long 31-hour circadian period previously observed in DGRP_892. We performed transcriptional profiling of whole fly heads from two genotypes: DGRP_892, and Canton-S B, a line with a normal 24-hour circadian period. We collected fly heads every two hours over a 24-hour period. We quantified differential expression among genotype, time, and sex. Overall design: We sequenced RNA from a Drosophila Genetic Reference Panel (DGRP) line with a long circadian period (DGRP_892) and a line with a 24-hour circadian period (Canton-S B). We collected 150 virgin male heads and 150 virgin female heads from each genotype every two hours over a 24-hour period to produce RNA sequence profiles. We reared fly cultures in two incubators 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. One incubator was set to a 6:00 am "lights on": 6:00 pm "lights off" schedule, while the second incubator was set on the reciprocal light:dark cycle of 6:00 pm "lights on": 6:00 am "lights off". Adult flies were entrained to their respective light:dark cycles for three days. Both groups of flies were transferred to a common incubator and then subjected to constant darkness for three days. Flies were flash frozen on dry ice every 2 hours and frozen at -80 deg. C until processed. Flies were decapitated on dry ice, and total RNA was extracted. This process was repeated three times to produce 144 samples (three biological replicates per genotype/timepoint/sex). Prior to library preparation, ERCC Spike-in Control Mix I was added to each sample as a control. PolyA RNA stranded libraries were prepared using Illumina TruSeq Stranded mRNA Sample Prep kits. Unique bar code adapters were applied to each library, and dual indices were used. We performed sequencing on an Illumina HiSeq4000 using version 4 chemistry to achieve a minimum of 20-25 million 125-bp read pairs. Reads were mapped to FlyBase release 6 version 01 of the Drosophila melanogaster genome and the ERCC sequences. Mapped reads were counted at the gene level.