title: M3_male
design: Briefly, one RNA pool of 5 livers (n=1), from each treatment and sex, with a minimum concentration of 40 ng/L, were subjected to a quality control, to determine RNA purity by measuring the ratio of absorbance at 260/280 nm on a NanoDrop (Thermo Fisher Scientiphic, USA);  integrity  and concentration were estimated using the Agilent 2100 BioAnalizer (Agilent Technologies, USA) and integrity was further confirmed by 1 % agarose gel eletrophoresis. RNA samples with a RIN (RNA integrity number) above 6.3 passed the quality control and were used for library construction and further sequencing. After quality control proceedings, RNA samples were then enriched using oligo(dT) beads and rRNA was removed from total RNA using the Ribo-Zero Kit (Illumina, USA). Subsequently, sequencing libraries were prepared using Novogene NGS RNA Library Prep Set. Briefly, Isolated mRNA was randomly fragmented by adding fragmentation buffer and cDNA synthesis was performed using mRNA template and random hexamer primers for the first strand, after which a custom second-strand synthesis buffer (Illumina, USA), as well as dNTPs, RNAse H and DNA polymerase I were added to start the second strand synthesis.. After end repair,  A-tailing,  and sequencing adapter ligation, were performed. In order to select cDNA fragments of approximately 250-300 bp, samples were sonicated and magnetic beads were used to select the target fragment size, after which UTP-containing second strand were removed through enzyme digestion. cDNA library was then completed, through size selection andPCR amplification.  After cDNA library completion, a preliminary quantification was performed using a Qubit 2.0 fluorometer (Termo Fisher Scientific, USA), followed by Agilent 2100 Bioanalyzer to test insert size, and lastly a Q-PCR to quantify the library effective concentration precisely. Quantified libraries, from each treatment and sex, were then independently subjected to sequencing (Illumina Novaseq 6000 paired-end (2x150)), generating an average of 35 million pair-end reads for each sample.