Pineal gland; day and night (Human)
Specimens
Pineal glands were obtained from NDRI (National Disease Research Interchange; Pennsylvania Medical Center, Philadelphia, PA). Two glands were from individuals who died at mid-day (11:00-13:00) and four from deaths occurring at mid-night (23:00-01:00). Samples were stored at -80C until use.
RNA Preparation
RNA was extracted from individual glands with Trizol (Invitrogen, Carlsbad, CA) and subsequently cleaned up using an RNeasy Micro Kit with on-column DNase treatment (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), respectively. RIN values ranged from 7.3-8.7.
RNA-Seq library preparation and sequencing
Ribosomal-depleted RNA was used to construct stranded libraries from 1 µg aliquots of total RNA using a TruSeq Stranded Total RNA Sample Prep Kit (Illumina cat. no. RS-122-2301) according to the manufacturer's instructions. The library insert sizes were approximately 170 bp. Unique barcode adapters were appended to each library. The pooled libraries were sequenced on a HiSeq 2500 (Illumina, San Diego, CA) using version 4 chemistry. This yielded an average of 81 million 126-bp read-pairs for each sample.
Bioinformatics methods
See bioinformatics methods for details.