Qubit 2.0 fluorometer qubit rna hs assay kit

Total RNA was quantified by Qubit 2.0 Fluorometer (Qubit RNA HS Assay Kit, Life Technologies), and RNA purity was measured by spectrophotometer (NanoDrop 2000c, ThermoFisher Scientic). Bluehead RNA was reverse transcribed in a Mastercycler Pro S thermal cycler (Eppendorf) with the following protocol: 37 °C (15 mins), 85 °C (5 s), 4 °C until removal. For spotty and kyusen, reverse transcription reactions were performed in a SureCycler 8800 (Agilent Technologies) with the following protocol: 25 °C (10 mins), 37 °C (120 min), 85 °C (5 mins), 4 °C until removal. Further details are provided in Table S2.

The tissues were homogenized using TissueLyser II (QIAGEN®) (Center for Neuroendocrinology, Department of Anatomy, University of Otago). Forebrain/midbrain and gonadal total RNA were extracted with TRI reagent (Invitrogen) using chloroform (forebrain/midbrain) or bromochloropropane (gonads) as the phase separation reagent. Samples were then DNase-treated (TURBO DNA-free Kit, Ambion) and total RNA-cleaned (NucleoSpin RNA XS columns, Macherey-Nagel). RNA integrity was assessed on an Agilent 2100 Bioanalyzer. Sex-changing gonads consistently showed RNA profiles with a strong peak of low molecular weight RNA, which possibly corresponds to massive 5S RNA expression in atretic ovaries and masks the 18S and 28S rRNA peaks used for calculating RNA integrity numbers (RIN). Such patterns were also observed in ovaries and intersex gonads of thicklip gray mullets, Chelon labrosus [68 (link)], and ovaries of protandrous sharpsnout seabream, Diplodus puntazzo [20 (link)]. Therefore, RIN values could not serve as useful measures of RNA integrity in these sex-changing gonads of bluehead wrasses. For brain RNA, samples with RIN values above 6.0 were used for RNA-seq. Total RNA concentration was measured by Qubit 2.0 Fluorometer (Qubit RNA HS Assay Kit, Life Technologies), and samples were diluted to 10 ng/μL.