Pineal gland ZT miRNA; day and night (Rat_miRNA)
Specimens
Animals and sample preparation: Sprague Dawley rats (Female, 180-250 g; Taconic Farms, Germantown, NY) were maintained on a 14:10 light:dark (L:D) cycle. Two pools of six pineal glands were prepared; the day pool was obtained at Zeitgeber time (ZT) 7 and the night pool at ZT 19. Euthanasia during the dark period collection was performed using a dim red light. Animal use and care protocols were approved by local ethical review and were in accordance with National Institutes of Health guidelines.
RNA Preparation
RNA extraction: Total RNA was purified in a manner to recover the small RNA component using miRVana columns (Invitrogen), according to the manufacturer's instructions. The RNA was subsequently treated with TURBO DNA-free DNase (Invitrogen) for 15 min at room temperature. RNA quality and quantity were assessed using a Bioanalyzer (Agilent Technologies, Wilmington, DE); RNA integrity number values were determined to be ≥9.
RNA-Seq library preparation and sequencing
Library preparation and sequencing: Library preparation was performed according to the Small RNA Sequencing Version 1.5 Alternative Protocol (Illumina, San Diego). In this procedure, pre-adenylated RNA adaptors are ligated to the 3′ end of the small RNAs, followed by ligation of a second RNA adaptor to the 5′ end. Reverse transcription was performed using a primer against the 3′ adaptor. The cDNA product was subsequently amplified by 15 PCR cycles. An ∼90 to 98 bp band, corresponding to an original small RNA size of 18 to 26 nucleotides, was gel-purified and applied to the sequencing flow cell cluster generation station. The flow cell was run on an Illumina GAII system for 35 cycles using the small RNA sequencing kit (Illumina). Library preparation and sequencing were done by the National Institutes of Health Intramural Sequencing Center. This yielded an average of 16.6 million reads for each sample.
Bioinformatics methods
See bioinformatics methods for details.
References
- Clokie SJ, Lau P, Kim HH, Coon SL, Klein DC. MicroRNAs in the pineal gland: miR-483 regulates melatonin synthesis by targeting arylalkylamine N-acetyltransferase. J Biol Chem. 2012 Jul 20;287(30):25312-24. doi: 10.1074/jbc.M112.356733. PMID: 22908386