GSM5592666: GL2, liver, control diet; Danio rerio; RNA-Seq
Whole livers were dissected out, frozen in liquid nitrogen, and transferred to a -80C freezer for long-term storage. RNA was isolated from tissue using RNeasy™ Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) per the manufacturer's instructions. RNA with an RNA Integrity Number (RIN) of 7.0 or above was used for sequencing library preparation. The Agilent SureSelect Strand Specific mRNA library kit as per the manufacturer's instructions (Agilent, Santa Clara, CA).  Library construction began with ribosome reduction using the NEBNext rRNA depletion kit for human/mouse/rat as described by the manufacturer (New England Biolabs, Ipswich MA).  The resulting RNA was randomly fragmented with cations and heat, which was followed by first strand synthesis using random primers with inclusion of Actinomycin D (2.4ng/µL final concentration).  Second strand cDNA production was done with standard techniques, the ends of the resulting cDNA were made blunt, A-tailed and adaptors ligated for amplification and indexing to allow for multiplexing during sequencing.  The cDNA libraries were quantitated using RT-PCR in a Roche LightCycler 480 with the Kapa Biosystems kit for Illumina library quantitation (Kapa Biosystems, Woburn, MA) prior to cluster generation.  Cluster generation was performed according to the manufacturers recommendations for onboard clustering (Illumina). Samples were sequenced to achieve a minimum of 30 million, single-end, 75bp reads per sample.