SRA

Purpose: We performed an NGS study on the circadian changes in human pineal gland transcriptome in order to elucidate novel and conserved elements in the circadian clock, as well as to conduct a comparative analysis of pineal transcriptomes of several animal species. Methods: Total RNA from human pineal glands of individuals that died at 2 timepoints (Mid-Day, Midnight) was deep sequenced, using Illumina HiSeq2500. Reads were aligned using STAR aligner and differential expression was asssessed using DESeq2. Results: We discover a variety of genes that show circadian activivty in human pineal gland. Conclusions: Our study represents part of a comparative analysis of pineal gland transcriptome of several species, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Overall design: Pineal glands were obtained from NDRI (National Disease Research Interchange), (Pennsylvania Medical Center, Philadelphia, PA). Two glands were from individuals that died at mid-day (11:00-13:00) and four from deaths occurring at mid-night (23:00-01:00). Samples were stored at -80 o C until use. 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 NanoDropspectrophotometer (NanoDrop, Wilmington, DE) and an Agilent 2100 Bioanalyzer (AgilentTechnologies, 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. Equal volumes of individual libraries were pooled and run on a MiSeq (Illumina, San Diego, CA). The libraries were then re-pooled based on the MiSeq demultiplexing results. The pooled libraries were sequenced on a HiSeq 2500 (Illumina, San Diego, CA) using version 4 chemistry. The data was processed using RTA version 1.18.64 and CASAVA 1.8.2. This yielded an average of 81 million 126-bp read-pairs for each sample. Reads were aligned using STAR aligner and differential expression was asssessed using DESeq2. GRCh37/hg19 genome build with Gencode 19 annotation was used.