male gonad of frog
Total RNA was isolated using Trizol Reagent (Invitrogen Life Technologies). A NanoDrop spectrophotometer (Thermo Fisher Scientific) was used to assess the concentration, quality, and integrity of samples. Sequencing libraries were created using a TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA). mRNA enrichment was performed using oligo-dT magnetic beads, followed by fragmentation of RNA. Reverse transcription was performed to synthesize the first and second cDNA strands. Finally, end repair was performed, an A-tail was added, and sequencing adapters were incorporated to complete the library preparation process. To select the preferred length of cDNA fragments (380 bp), library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA). DNA fragments with ligated adaptor molecules at both ends were selectively enriched using the Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. The products were purified (AMPure XP system) and quantified using an Agilent High-Sensitivity DNA Assay on a Bioanalyzer 2100 system (Agilent Technologies). The library was sequenced on a NovaSeq 6000 platform (Illumina) by Shanghai Personal Biotechnology Cp. Ltd. Sequencing data were processed using FASTP (version 0.22.0) to filter and obtain high-quality sequences, referred to as Clean Reads, for subsequent analyses. Next, Trinity (version 2.15.1) was used to assemble the Clean Reads into transcripts for further examination. Upon completion of the assembly process, transcript sequence files were generated in FASTA format. The longest transcript for each gene was designated as a representative sequence and termed a unigene.