Tripterygion delaisi SRA sample
Fish brains were dissected out of the frozen heads, weighed and placed in TRI Reagent. All T. delaisi brains weighed between seven and twenty milligrams. Tissues were homogenized in a Retsch homogenizer (MM200) at 25Hz for two intervals of 30 seconds. Phase separation was done with BCP Phase Separation Reagent (1-Bromo-3-Chloropropane) and all centrifuge steps were performed at 4oC. The RNA was precipitated with isopropanol and the total RNA pellet was washed twice with 75% ethanol to then be dissolved in TE Buffer pH 8.0.  Poly-A mRNA was purified using Dynabeads (Invitrogen) coated in Oligo(dT)25 following the manufacturer’s protocol, but adding a second wash-step. First-strand cDNA was synthesized with Random Hexamer Primers and SuperScript reverse transcriptase and second strand cDNA synthesis was performed using RNaseH and DNA Polymerase I. cDNA was then purified with the QIAquick PCR purification kit and fragmented with dsDNA fragmentase for 28 minutes at 37oC to yet again be purified with the purification kit. Fragments were prepared for Illumina sequencing with New England Biolabs reagents following the manufacturer’s instructions for each reagent module. Firstly, ends were repaired with the End Repair module using E.coli ligase and End Repair enzyme mix. A single A-base was then added using the dA-Tailing module with Klenow DNA polymerase. Homemade four-mer barcodes were added with help of the Quick T4 DNA ligase to be able to multiplex the samples in the same Illumina lane. After each step the DNA was purified either with the PCR purification kit or the Qiagen MinElute PCR purification kit. Subsequently, 200-300bp fragments were gel purified in Invitrogen ultra-pure agarose and cleaned with the QIAquick gel extraction kit. Finally, cDNA fragments were enriched using Phusion polymerase for 15 cycles and once more purified with the PCR purification kit with a final volume of 30µl. Concentration and purity was measured several times throughout the process with the Agilent 2100 Bioanalyzer (Agilent RNA 6000 pico & DNA 1000 Kit).
University of Barcelona
Tripterygion delaisi Dominant Male 3