Pineal gland and retina; time series; constant darkness (Chicken)
Specimens
One-day-old white leghorn chickens (Gallus domesticus) were entrained to a 12h light: 12h dark cycle of illumination (L:D 12:12) for 9 days. The light cycle was switched to L:D 0: 24 (constant darkness) on day 10. Sampling was initiated immediately at the beginning of the subjective day. Pools of three pineal glands or single retinas were collected in triplicate (rep1, rep2, rep3) at circadian time (CT) 0, 4, 8, 12, 16, 20. Total RNA was extracted using TRIzol, followed by clean-up with an RNeasy Kit with on-column DNase treatment.
RNA Preparation
The RNA-Seq libraries were constructed using Illumina TruSeq RNA sample preparation kit (San Diego, CA). Total RNA (500 ng per sample) spiked with ERCC Mix1 or Mix2 was polyA-selected and fragmented. Samples were bar-coded such that each sample of a triplicate received a different bar-code; 36 total combinations. To assess the quality of the libraries, twelve different libraries derived from 12 different RNA samples (2 tissues x6 time points) were loaded in a single lane of Illumina's TruSeq V3 flow cell (total 3 lanes). The quality of the libraries was also checked using an Agilent Bioanalyzer DNA 1000 chip and each cDNA library was quantified by Qubit 2.0 Fluorometer (Life Technologies, Grand Island NY). The sequencing was carried out using an Illumina HiScanSQ with 12 different libraries in each lane (see above), repeated in 2 lanes, yielding an average of 35 million uniquely-aligned, paired-end, 100 bp reads per sample.
Bioinformatics methods
See bioinformatics methods for details.