Male gonads were collected into 2 ml tubes and stored at − 80 °C freezer. Samples were homogenized using TissueLyser LT (Qiagen) for 10 min at 50 Hz. Total RNA was isolated using 1 ml of TRI Reagent (Sigma-Aldrich, T9424) following the manufacturer's manual. The RNA pellet was then dissolved in 40 μl DNAse solution (32 μl Nuclease-free water, 4 μl 10x reaction buffer, 4 μl DNAse I, Sigma-Aldrich, AMPD I), and incubated at 37 °C for 30 min. Afterwards, 40 μl of 8 M LiCl (Sigma-Aldrich, L7026) were added for RNA precipitation. This solution was incubated overnight in − 20 °C freezer and then centrifuged for 30 min at 16,000 g. The supernatant was removed, and the RNA was then washed twice with 1 ml of 80% ethanol followed by centrifugation for 30 min. The final total RNA was diluted in 20 μl of 1xTE buffer (Invitrogen, 12,090–015). The concentration of RNA was measured using Nanodrop 8000 (Thermo Scientific), and the quality of RNA was analysed using Fragment Analyzer (AATI, Standard Sensitivity RNA analysis kit, DNF-471). Only samples with RQN higher than 6.9 were used for the following analysis. SureSelect Strand-Specific mRNA library protocol was used for library preparation with 360 ng mRNA as input. All steps were according to the manufactory manual with one exception; RiBO cop Select and Deplete rRNA Depletion Kit V1.2 (Lexogen) was used for rRNA depletion instead of poly-A method. Final libraries were pooled and controlled with DNF-474-0500 HS NGS Fragment kit (1-6000bp) using Fragment Analyzer (AATI) according to the manual.