Instrument: Ion Torrent PGM
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Larval medaka were euthanized at 20 dpf in 0.125% tricaine/ERM solution and replicates were pooled to ensure 15-25 individuals per RNA sample. Specimen were transferred to Leibowitz’s media (L-15) containing 10% Fetal Bovine Serum. Dechorionated embryos or hatched larvae were passed through a 20-22 gauge hypodermic needle and 3-ml syringe several times to isolate the axial skeleton from craniofacial and abdominal viscera. Sheared tissue was collected on 105-μm nylon mesh and transferred to fresh L-15/10% FBS media. Under a dissecting light microscope axial tissue was inspected and trimmed of any remaining craniofacial or abdominal tissue. Samples were immediately flash frozen in liquid nitrogen. Tissues were homogenized in TRI Reagent® and total RNA was isolated according to the TRI Reagent manufacturer’s protocol. Finally, total RNA was quantified using Agilent 2100 Bioanalyzer and 2100 Expert Software package (Agilent Technologies). RNA samples with RNA Integrity numbers (RINs) of 9.9 or greater were used for RNA-Seq library generation. Whole transcriptome libraries were prepared using Ion Total RNA-Seq Kit v2 (Ion Torrent, Life Technologies, Carlsbad, CA) for the Ion PGM sequencing platform as described by the manufacturer. Briefly, polyA mRNA was purified for each sample from 2 μg of denatured total RNA with oligo(dT)25 Dynabeads®. Adaptors from Ion Adaptor Mix v2 were ligated to poly(A) RNA, and each sample was reverse transcribed to form cDNA, and then amplified using Platinum® PCR Supermix High Fidelity, Ion Xpress™ RNA 3’ Barcode Primers, and a different Ion Xpress® RNA-Seq BC Primer (BC#11-16) for each sample to form a barcoded library. The library was then pooled with equimolar amounts from each of the 6 barcoded samples. The pooled library was amplified of the pooled library using the Ion OneTouch™ 200 and Ion PGM Template OT2 200 Kit. The solution containing template-positive ISPs was enriched on the Ion OneTouch™ ES according to the manufacturer’s protocol. Sequencing primers were annealed to the template-positive ISPs, Ion PGM™ Sequencing 200 v2 Polymerase was added to the ISP solution, and incubated at room temperature for 5 minutes. Finally, the template-positive ISPs + Sequencing Polymerase mixture were carefully loaded into the Ion 318™ chip and sequenced overnight on the Ion PGM™ System.