Total RNAseq from fly heads at different ages - circRNAs are extremely stable in the brain and can serve as age and experience markers [CSaging]

Circular RNAs are a large class of stable RNAs generated by backsplicing that accumulate with age in neural tissue. To determine the reason for this accumulation, we profiled circRNAs from fly heads at six timepoints during their lifespan. We found that the global levels of circRNAs increase linearly with age and that circRNAs are excellent age-markers. At the individual level, we found that most circRNAs increase their levels with age, and this increase does not correlate with changes on overall transcription, the levels of the mRNA counterparts, changes in intron retention, or splicing of the host gene, demonstrating that the increase is not due to biogenesis. In addition, a sustained stimulus, like transient exposure to higher or lower temperatures, leads to increases (but no decreases) in the levels of specific circRNAs further demonstrating the stability of these molecules in vivo. This unusually high stability suggests that circRNAs can be used as experience markers. Indeed, flies that were treated for ten days at 29°C display high levels of specific temperature-induced circRNAs even six weeks after being returned to standard conditions. Together, our data demonstrates that circRNAs are extremely stable in vivo and that they can be utilized as stress/life experience markers. Overall design: CantonS, wild-type, flies were grown at 25C in 12:12 light:dark conditions, allowed to mate for 3 days prior to separating males and females for aging. After that, flies were flash frozen in liquid nitrogen 0, 10, 20, 30, 40, or 50 days later. Samples were collected in duplicate or triplicates. RNA from the fly brains was extracted using TRIzol reagent (Sigma, T9424) and treated with DNaseI (NEB, M0303L). We used 1 ug of RNA as input for ribodepletion (with a protocol based on Adiconis et al. 2013). Total RNA libraries were prepared with NEXTFLEX Rapid Directional RNA-Seq Kit 2.0 (PerkinElmer) as recommended by the manufacturer. Samples were sequenced by Novogene (Novogene Corporation Inc 8801 Folsom BLVD, Suite 290, Sacramento, CA) with HiSeq-4000.