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. Female Sprague Dawley rats (Taconic Farms, Germantown, NY) were used for these studies and were maintained on a 14:10 light:dark (L:D) cycle before being euthanized (2 to 3 months old). Pineal glands were removed and cultured as described (1) for a period of 48 hours under control conditions, then treated with norepinephrine (NE; 1uM) or dibutyryl cyclic AMP (DBcAMP; 500 uM), or left untreated (CN) for 6 hours before being harvested into microtubes placed on solid CO2 (three glands per tube). For each of the three treatment conditions, samples were pooled into three biological replicates (9 pooled samples, total 27 glands)

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 175bp. Unique barcode adapters were applied to each library. Equal volumes of individual libraries were pooled 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 two lanes on a HiSeq2000 (Illumina, San Diego, CA) using version 3 chemistry. This yielded an average of an average of 43.0 million read-pairs per sample (ranging from 39.8 million to 49.7 million).

Bioinformatics methods

See bioinformatics methods for details.

References

1. Parfitt A, Weller JL, Klein DC. Beta adrenergic-blockers decrease adrenergically stimulated N-acetyltransferase activity in pineal glands in organ culture Neuropharmacology. 1976;15(6):353-8. Epub 1976/06/01.

2. 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