Pineal gland, retina and mixed tissue; time series (Rhesus)
Specimens
Animals: Rhesus macaques were obtained through the Veterinary Resources Program Primate Recycle Program of NIH over a period of 11 years. Eighteen adults ( 3 to 33 years of age, male and female, 4 to 10.3 kg body weight) were used. All protocols and handling of the animals conformed to the Guidelines for Care and Use of Laboratory Animals (NIH Publication 80-23; Protocols 99-012 and 11-055). Animals were housed under a 12-h light, 12-h dark lighting regimen (lights on at 0600 h) in NIH facilities for at least 6 weeks. The light level in the cages was approximately 325 Lux. Twelve animals (three per time point) were sacrificed between 11:00 and 13:00 h (day), 17:00 h and 19:00 h (dusk), 23:00 h and 01:00 h (night) and 05:00 h and 07:00 h (dawn). Animals were anesthetized (ketamine, 0.1 mL/kg body weight) and a catheter was placed in the saphenous vein prior to transport to the procedure room. Animals were sacrificed using Euthasol (Virbac, Fort Worth, TX) or Beuthanasia D (Schering Plough, Union, NJ). Animals sampled at night or dusk were anesthetized under dim red light and blindfolded; euthanasia was done under dim white light and the blindfold was removed after decapitation. In addition to these twelve animals, tissues were obtained from six other animals to prepare RNA used for a mixed RNA sample. Removal of tissues, including the pineal gland, retina, and others (corpus callosum, frontal cortex, cerebellum, thalamus, hippocampus, caudate, medulla, pituitary, heart, liver, lung, kidney, skeletal muscle, small intestine, testis and ovary) was complete within 45 minutes following euthanasia; immediately following removal, samples of each tissue were placed on solid CO2. Tissue was stored at -80C.
RNA Preparation
RNA extraction: Total RNA was extracted from individual pineal glands or pieces of other tissues (2 to 4 mm3 pieces) with TRIzol reagent (Invitrogen, Carlsbad, CA), followed by clean-up using an RNeasy Micro Kit with on-column DNase treatment as per the manufacture's protocol (Qiagen). The amount and quality of RNA were determined using a NanoDrop spectrophotometer (NanoDrop, Wilmington, DE) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). RIN values were > 9 for all tissues. Two mixed tissue RNA samples were prepared (Day, male; Night, female) by adding equal amounts of RNA extracted from sixteen tissues, not including the pineal gland or retina. All RNAs in a sample originated in tissues obtained from a single animal, except for RNA from testis and ovary, which were obtained from additional animals.
RNA-Seq library preparation and sequencing
RNA-Seq libraries were constructed from 0.7-1ug total RNA using the TruSeq Stranded Total RNA Sample Prep Kits (Illumina cat. no. RS-122-2301) following the manufacturer's instructions. Insert sizes were approximately 175bp. Unique barcode adapters were applied to each library. Equal volumes of individual libraries were pooled and run on a MiSeq instrument (Illumina, San Diego, CA). The libraries were then repooled based on the MiSeq demultiplexing results. The pooled libraries were sequenced on a HiSeq2000 (Illumina, San Diego, CA) using version 3 chemistry.
Bioinformatics methods
See bioinformatics methods for details.