RNA-Seq of Drosophila tim promoter/enhancer mutants around the clock

The contribution of cis-regulatory elements to Drosophila circadian gene expression is poorly understood. We generated a series of CRISPR-mediated deletions within the regulatory regions of the circadian gene timeless (tim) and characterized them through multiple high-throughput sequencing experiments. We isolated heads from wild-type and mutant flies around the clock and performed RNA-Seq on them to determine the effects of regulatory element deletions on circadian gene expression. Overall design: Male and female flies were entrained in 12:12 LD conditions for 4 days at 25°C, then collected at ZT2, ZT6, ZT10, ZT14, ZT18 and ZT22. Total RNA from fly heads was extracted using TRIzol reagent (Invitrogen) following the supplier's protocol. The resulting RNA was used to make libraries for transcriptome sequencing using the TrueSeq RNA Sample Prep Kit (v2; Illumina). The quality of the libraries was assessed using a Bioanalyzer 2100 (Agilent) and then sequenced on a NextSeq 550 (Illumina). RNA libraries were mapped to Drosophila genome dm6 using STAR with default setting and expression levels were quantified using FeatureCounts. Two independent biological replicates were done with the exception of yw at ZT18.