Specimens

Experimental design: These data were generated in three parts. In the first, submitted in 2012 for the Tovin et al., 2012 paper, adult (0.5–1.5 years old) transgenic zebrafish, Tg(aanat2:EGFP) Y8, 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, and transferred to constant darkness (DD) for tissue collection. Starting from circadian time (CT) 14, pineal glands were collected at 4-hr intervals for 48 hours (12 time points identified as CT 14, 18, 22, 2, 6, 10, 14b, 18b, 22b, 2b, 6b and 10b). Pools of 20 pineal glands were prepared at each time-point and total RNA was extracted using the RNeasy Lipid Tissue Mini Kit (QIAGEN), according to the manufacturer's instructions. The Illumina TruSeq protocol was used to prepare libraries from RNA samples.

In the second, submitted in 2013 (unpublished), the same fish, culture procedure (sampled during DD), sample preparation and sequencing described above were used. Triplicate samples of pineal glands were collected during the subjective light (CT6) and dark phases (CT18). Pools of 20 pineal glands were prepared at each time-point and total RNA was extracted using the RNeasy Lipid Tissue Mini Kit (QIAGEN), according to the manufacturer's instructions. The Illumina TruSeq protocol was used to prepare libraries from RNA samples.

In the third, submitted in 2016 for Levin et al., 2016, adult homozygous Tg(aanat2:EGFP-ΔCLK)(abbreviated as deltaCLK) zebrafish were cultured and sampled under the same conditions described above so that the daily rhythms of the two strains could be compared. To confirm that results would be comparable, Tg(aanat2:EGFP) Y8 fish were also sampled. Fish were raised in a temperature-controlled recirculation water system under 12-hr:12-hr LD cycles, and transferred to DD at the end of the light period prior to sampling. Pineal glands were sampled at 4-hr intervals throughout two daily cycles under DD at 12 time points corresponding to circadian time (CT) 14, 18, 22, 2, 6, 10, 14b, 18b, 22b, 2b, 6b and 10b. A pool of 16 pineal glands was collected at each time point. In addition, two control pools of 14 pineal glands were collected from Tg(aanat2:EGFP) fish at time points corresponding to CT2 and CT14b. Fish were anesthetized in 1.5 mM Tricane (Sigma), sacrificed by decapitation, and pineal glands were removed under a fluorescent dissecting microscope. Total RNA was extracted using the RNeasy Lipid Tissue Mini Kit (QIAGEN), according to the manufacturer's instructions.

RNA-Seq library preparation and sequencing

For the first analysis, overall, 12 libraries (12 time points) were run on 2 lanes of an Illumina HiSeq2000 machine using the multiplexing strategy of the TruSeq protocol (Institute of Applied Genomics, Italy). On average, ∼30 million paired-end reads were obtained for each library. The reads were 2×100 base pairs for 8 time points (CTs 22, 2, 6, 10, 18b, 2b, 6b, 10b) and 2×50 base pairs for the remaining time points (CTs 14, 18, 14b and 22b).

For the second, 6 libraries (3 light groups and 3 dark groups) were run on one lane of an Illumina HiSeq2000 machine using the multiplexing strategy of the TruSeq protocol (Institute of Applied Genomics, Italy). On average, ~6.7 million paired-end reads were obtained for each library. The reads were 2×100 base pairs.

For the third, overall, 14 libraries [12 time points from Tg(aanat2:EGFP-ΔCLK) fish and two control samples from Tg(aanat2:EGFP) fish] were run on a single flow cell of an Illumina HiSeq2500 machine (Rapid Run mode) using the multiplexing strategy of the TruSeq protocol (Institute of Applied Genomics, Italy). On average, 14 million paired-end reads were obtained for each library. The reads were 2×50 base pairs.

All three parts are included together in a single NCBI Short Read Archive (SRA) entry. This SRA entry is referenced in Tovin et al., 2012 as SRA054264. The same entry is referenced in Livne et al., 2016 as SRP016132.

Bioinformatics methods

See bioinformatics methods for details.

References

(1) Tovin A, Alon S, Ben-Moshe Z, Mracek P, Vatine G, Foulkes NS, Jacob-Hirsch J, Rechavi G, Toyama R, Coon SL, Klein DC, Eisenberg E, Gothilf Y. Systematic identification of rhythmic genes reveals camk1gb as a new element in the circadian clockwork PLoS Genet. 2012;8(12):e1003116. doi: 10.1371/journal.pgen.1003116. Epub 2012 Dec 20.

(2) Ben-Moshe Livne Z, Alon S, Vallone D, Bayleyen Y, Tovin A, Shainer I, Nisembaum LG, Aviram I, Smadja-Storz S, Fuentes M, Falcón J, Eisenberg E, Klein DC, Burgess HA, Foulkes NS, Gothilf Y. Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior. PLoS Genet. 2016 Nov 21;12(11):e1006445. doi: 10.1371/journal.pgen.1006445. eCollection 2016 Nov.