GSM4187015: 3dpRFed1Gut2; Danio rerio; RNA-Seq
Frozen intestinal samples stored in Trizol at -80oC were homogenized using Zirconium oxide beads (Biospec, 11079107) and a Vortex Genie2 (Scientific Industries, 1311-V) fitted with a Vortex Adapter (Scientific Industries, 13000-V1-24) in three 45-second intervals. Samples were put on ice in-between homogenization to prevent overheating.  Following homogenization, a phase separation was performed by adding 200ul of chloroform to each sample and mixing by vigorous inversion 15 times. Samples were then incubated at room temperature for 3 minutes and centrifuged at 12000rcf for 15min at 4oC. 500ul of the aqueous upper phase from each sample was then transferred to a new Eppendorf tube, to which 500uL 70% Ethanol in DEPC water was added and vortexed. Following phase separation, samples were DNase treated and total RNA was extracted via column purification using the PureLink DNase Set (Thermo Fisher, 12185010) and the PureLink RNA Mini kit (Thermo Fisher, 12183025) according to the manufacturer's instructions. Final sample quality and concentration were assessed via spectrophotometry and samples were stored at -80oC until submission to the Duke Sequencing and Genomic Technologies Core. RNA-seq libraries were prepared and sequenced by Duke Sequencing and Genomic Technologies Core on an Illumina HiSeq 2500 (50bp single-end read) according to the manufacturer's instructions. Prior to library preparation, RNA was extracted using the Ribozero Gold Kit (Cat #20020598) according to the manufacturer's instructions.