Specimens

Animals and sample preparation: Animal use and care protocols were approved by the NIH Institutional Animal Care and Use Committee and followed the guidelines of the National Research Council's Guide for Care and Use of Laboratory Animals (Vol. 8) and the Animal Research: Reporting In vivo Experiments (ARRIVE) guidelines. Sprague Dawley rats (Taconic Farms, Germantown, NY) were used for these studies and were maintained on a 14:10 light:dark (L:D) cycle. Pineal gland samples were taken at mid-day (ZT7) or midnight (ZT19). Additional samples were taken from the following tissues from 3 animals at ZT7: cortex, cerebellum, midbrain, hypothalamus, hindbrain, spinal cord, retina, pituitary, heart, liver, lung, kidney, skeletal muscle, small intestine, and adrenal gland. Pineal glands were immediately placed in microtubes on solid CO2 (three glands per tube); samples of other tissues (approximately 10 mg) were rapidly prepared and placed in individual tubes. Tissues were stored at -80C until use. Total RNA was extracted from each tissue, and then equal amounts of each of the 15 tissues were combined for the final pooled 'mixed-tissue' sample. Three pooled samples were created: one each for pineal-day, pineal-night, and mixed-tissue (3 pooled samples, total).

RNA Preparation

RNA extraction: Total RNA was extracted 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, Valencia, CA). 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). Each pool yielded 2.1 to 3.7 micrograms of total RNA (RIN values > 9).

RNA-Seq library preparation and sequencing

Library preparation and sequencing: Stranded RNA-Seq libraries were constructed from 0.7-1 ug total RNA using the TruSeq Stranded Total RNA Sample Prep Kits (Illumina cat. no. RS-122-2301) according to manufacturer's instructions. The library insert sizes were approximately 270bp. Unique barcode adapters were applied to each library. Equal volumes of individual libraries were pool and run on a MiSeq (Illumina, San Diego, CA). The libraries were then repooled based on the MiSeq demultiplexing results. The pooled libraries were sequenced on 1 lane of a HiSeq2000 (Illumina, San Diego, CA) using version 3 chemistry. These samples were indexed and pooled and run on a single lane. This yielded an average of 62.8 million read-pairs per sample for the pineal marker gene samples (ranging from 55.6 million to 69.1 million). Analysis and listing of pineal marker genes are given in Hartley et al., 2015.

Bioinformatics methods

See bioinformatics methods for details.

References

1. Hartley SW, Coon SL, Savastano LE, Mullikin JC; NISC Comparative Sequencing Program, Fu C, Klein DC. Neurotranscriptomics: The Effects of Neonatal Stimulus Deprivation on the Rat Pineal Transcriptome PLoS One. 2015 Sep 14;10(9):e0137548. doi: 10.1371/journal.pone.0137548. eCollection 2015