RNA-Seq of nr5a1a and nr5a1b mutant zebrafish

Zebrafish were euthanized in Tricaine. We isolated gonad-containing trunk of theanimals by removing the anterior of the fish from just posterior of the pectoral finand removing the caudal peduncle posterior to the anus. Trunks were individuallyhomogenized in 200ul Trizol. Total RNA was extracted using the Ribopure RNAPurification Kit (ThermoFisher). Total RNA was enriched for mRNA using Dynabeads(r)Oligo(dt)25 (ThermoFisher). We constructed indexed, strand-specific cDNA sequencinglibraries using the NEXTflex(tm) qRNA-seq kit (BIOO Scientific). Libraryconcentrations were quantified using a Qubit(r) fluorometer (Life Technologies),normalized to a concentration of 2.3nM, and multiplexed. Prior to sequencing, wefurther evaluated the quality of the multiplexed library by quantitative real-timePCR using the Kapa Library Quantification Kit (Kapa Biosystems). Two lanes ofpaired-end 150 base pair (bp) sequencing were performed on an Illumina HiSeq 4000.