Oligo dt beads kit

Six gonad samples (three replicates each sex) were used for the preparation of transcriptome sequencing libraries. The RNA-Seq process was performed as described previously [24 (link)]. In brief, total RNA was isolated from female and male D. hystrix gonad tissues using a Trizol reagent kit (Life Technologies, Carlsbad, CA, USA). The isolated RNA was quantified by a Nanodrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and its integrity was confirmed by agarose gel electrophoresis and Agilent 2100 BioAnalyzer System (Agilent Technologies, Santa Clara, CA, USA). After purifying mRNA with an Oligo-dT Beads Kit (Qiagen, Hilden, Germany), cDNA libraries were constructed using a TruSeq^® Stranded mRNA Sample Preparation kit following the manufacturer’s protocol. RNA sequencing of the libraries was performed using the Illumina HiSeq™ 2000 platform (Illumina, Inc., San Diego, CA, USA) that generates paired-end (PE) reads of 125 bp length.

Total RNA was extracted from each tissue of both female and male B. splendens using the Trizol kit (Life Technologies, Carlsbad, CA, USA). The extracted RNA was treated by DNase I (TaKaRa Biotech Co., Ltd., Dalian, China) to eliminate genomic DNA. The concentration of total RNA was detected by NanoDrop2000c (Thermo Scientific, Wilmington, DE, USA), utilizing the absorbance at 260 nm, and the purity was assessed by OD_260/280 (acceptable range, 1.8–2.0). The 18S and 28S ribosomal bands stained with ethidium bromide on 0.8% agarose gels were used for the assessments of RNA integrity. After collecting the same amount of RNA samples from different tissues (about 1 μg for each tissue), both male and female RNA samples (about 5 μg for each sex) were sent to Guangzhou Jinyu Biotechnology Co., Ltd. (Guangzhou, Guangdong, China) for cDNA library construction and sequencing. The Oligo-dT Beads Kit (Qiagen, Hilden, Germany) was used to purify mRNA and cDNA libraries were constructed according to the Illumina RNA sequencing protocol. Two cDNA libraries were sequenced on an Illumina HiSeq™ 2000 sequencing platform (Illumina, Inc., San Diego, CA, USA) and paired-end (PE) reads with a length of 125 bp were generated.