Ocess was performed as described previously [24]. In short, total RNA was isolated from female and male D. hystrix gonad tissues making use of 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). Right after purifying mRNA with an Oligo-dT Beads Kit (Qiagen, Hilden, Germany), cDNA libraries were constructed utilizing a TruSeqStranded mRNA Sample Preparation kit following the manufacturer’s protocol. RNA sequencing with the libraries was performed utilizing the Illumina HiSeqTM 2000 platform (Illumina, Inc., San Diego, CA, USA) that generates paired-end (PE) reads of 125 bp length. 2.4. De Novo Assembly By signifies of SOAPnuke (version 1.five.0) [25], the raw reads were pruned employing the software’s high-quality handle together with the parameters “-l ten -q 0.5 -n 0.05 -p 1 -i”. Within this step, clean data have been generated by removing adapter sequences, reads containing ploy-N sequences and low-quality reads in the raw data. Then, the clean data have been de novo assembled by Trinity RNA-Seq Assembler (version r20140717, http://trinityrnaseq.sourceforge.net (accessed on 15 June 2015)) with default parameters [26]. The shorter redundant final linear transcripts have been eliminated employing CD-HIT-EST when the sequences have been totally covered by other transcripts with 100 identity, plus the longest ones had been defined as unigenes [24].Animals 2021, 11,four of2.five. Annotation and Classification Annotation was carried out by aligning sequence information against public databases utilizing BLAST 2.two.26+ software (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 20 April 2016)) with an E-value threshold of 1e-5. The unigenes had been subjected towards the sequence homology searches against the National Center for Biotechnology Facts (NCBI) non-redundant (Nr), Protein household (Pfam), Clusters of Orthologous Groups of proteins (KOG/COG/eggNOG), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Additional evaluation was performed to obtain the Gene Ontology (GO) functions making use of the Blast2GO PI3Kα supplier package [27]. The classification of GO terms was visualized utilizing WEGO statistical application [28]. Moreover, KOBAS v2.0 (http://kobas.cbi.pku.edu.cn/home.do (accessed on 24 July 2015)) was employed to analyze the KEGG pathway annotation information and to obtain the pathway categories [29]. two.six. Differential Expression Evaluation and Plasmodium site Functional Enrichment By suggests on the anticipated number of fragments per kb per million reads (FPKM) technique, gene expression levels have been calculated using RSEM software program (version 1.two.15) [30]. The DESeq2 package was utilized to recognize differentially expressed genes (DEGs) in between ovaries and testes [31]. FDR worth 0.01 and |log2 (Fold Change)| 1 had been applied because the threshold for significantly differential expression. Also, GO and KEGG functional enrichment analyses were performed to identify the DEGs that were significantly enriched in GO terms and KEGG pathways at Bonferroni-corrected p-value 0.05 compared with the whole-transcriptome background. GO enrichment analysis of DEGs was implemented by the topGO package’s (version two.28.0) Kolmogorov mirnov test [32]. Ultimately, KOBAS v2.0 was used to test the statistical enrichment of DEGs in KEGG pathways [33]. 2.7. Validation of DEGs by Real-Time Quantitative PCR (RT-qPCR) A total of 23 DEGs p.
