title: GSM2695596: OHara_sample_113; Eumetopias jubatus; RNA-Seq
library_construction_protocol: Whole blood collected in PaxGene™ RNA tubes were placed in a cooler with ice until transport back to the ship, at which point they were kept in the freezer at approximately -15°C. Upon return to UAF the samples were placed in a -80°C freezer and then shipped to the University of Washington Center for Exposures, Diseases, Genomics and Environment in insulated shipping containers with dry ice and subsequently stored at -80°C until extraction. Total RNA was extracted using the PaxGene™ Blood RNA Kit according to the manufacturer’s protocol. RNA integrity was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California).  RNA quantity was determined by measuring OD260 with a Thermo Scientific NanoDrop™ 1000 Spectrophotometer (Thermo Fisher Scientific, Inc.; Wilmington, DE). Total RNA is quantified using the Quanti-iT RNA Assay Kit and RIN scores are confirmed using the Agilent 2100 Bioanalyzer.  A Perkin Elmer Janus workstation is used to normalize library construction input to 1ug per sample.  Next-generation sequencing libraries are prepared in an automated, high-throughput format using the TruSeq Stranded mRNA kit.  All the steps required for library construction have been automated and are performed on a Sciclone NGSx Workstation. During library construction, ribosomal RNA is depleted by means of poly-A enrichment and first and second strand cDNA syntheses are performed.  Each library is then uniquely barcoded using the Illumina adapters and amplified using a total of 13 cycles of PCR. After amplification and cleanup, library concentrations are determined using the Quant-iT dsDNA High Sensitivity Assay.  Library fragment lengths are confirmed using the Agilent 2100 Bioanalzyer.  Barcoded libraries are subsequently normalized and pooled based on quantification values.  Clustering, followed by massively parallel sequencing-by-synthesis with fluorescently labeled, reversibly terminating nucleotides are carried out on the NextSeq instrument, achieving PE100 reads on a High Output flow cell.  Base calls are generated in real-time by the instrument.  Our read processing pipeline demultiplexes the reads and fastq files are produced by Picard.
attributes.tag: GEO Accession
attributes.value: GSM2695596