Next Generation Sequencing Study of Circadian Changes in Transcriptome of Zebrafish Pineal Gland and Eye

Purpose: We performed an NGS study on the circadian changes in zebrafish eye and pineal gland transcriptome in order to elucidate novel and conserved elements in the circadian clock. Methods: Poly-A selected RNA from zebrafish eyes and pineaal glands of animals eutanized at 2 timepoints (Mid-Day, Midnight) was deep sequenced, using Illumina HiSeq2000. Reads were aligned using STAR aligner and differential expression was asssessed using DESeq2. Results: We discover a variety of genes that show circadian activivty in both eye and pineal gland of Zebrafish. Conclusions: Our study represents part of a comparative analysis of retinal(eye) and pineal gland transcriptome of several species, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Overall design: Adult (0.5-1.5 years old) transgenic zebrafish, Tg(aanat2:EGFP), which express enhanced green fluorescent protein (EGFP) in the pineal gland under the control of the aanat2 regulatory regions, were used. Fish were raised under 12-hr light:12-hr dark (LD) cycles in a temperature controlled room. For tissue collection fish were anesthetized in 1.5 mM Tricane (Sigma), and sacrificed by decapitation. Dissections were conducted at mid-day (ZT6) and mid-night (ZT18). Fluorescent pineal glands, whole eyes and other tissues were removed under a dissecting microscope. A pool of 18 pineal glands 6 eyes and 4-8 of each peripheral tissue was collected at each time point. Total RNA for mRNA analysis was isolated using RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer''s instructions. The mixed tissue samples consisted of equal amounts of total RNA (1mg) from muscle, ovary, kidney, gill, liver, intestines, heart and brain. Poly-A selected RNA from zebrafish eyes, mixed tissue and pineaal glands of animals eutanized at 2 timepoints (Mid-Day, Midnight) was deep sequenced, using Illumina HiSeq2000. Reads were aligned using STAR aligner and differential expression was asssessed using DESeq2.

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