DOI: 10.6084/m9.figshare.7138280.v1
title: Differential gene expression analysis

description: <b>Shrimp strains and tissue collection</b> The red, yellow and transparent strains were obtained from Xiamen Fu shrimp Agricultural Development Co., Ltd., Xiamen, China. All three strains were cultured in plastic aquarium tanks (30×30×45 cm). Approximately 200 adults were kept in each plastic aquarium tank, and were feed with ornamental shrimp feed (Shirakura, Japan) twice a day at 08:00 and 17:00 h. About 50% water change was carried out once per three days. Few main water quality parameters remained stable (water temperature 25℃, dissolved oxygen 7.0-8.0mg/L, pH 7.0-7.4). With the help of anatomy microscope, the integument of the second abdominal segment was stripped on ice. Other tissues were removed using 0.7% normal saline solution. The 10 individuals’ integuments of the same strain were mixed together. The mixture was washed quickly with 0.7% normal saline, blotted on filter paper, put into liquid nitrogen, and then stored at -80℃ until RNA extraction was performed. There were 9 mixtures from each strain. <b>RNA preparation, cDNA library and and Illumine RNA-seq</b> Total RNA was prepared from a mixture of 10 individuals using TRIzol reagent (Invitrogen, USA) according to the manufacture’s protocol. RNA degradation and contamination were determined using 1% agarose gel electrophoresis. RNA concentration and purity were checked using a Nano-Photometer spectrophotometer (IMPLEN, CA, USA), and RNA integrity was assessed using an RNA Nano 6000 Assay Kit that was provided with a Bio-analyzer 2100 system (Agilent Technologies, CA, USA). Equal amounts of total RNA from 3 mixtures of the same strain was mixed to a pool. There were 3 RNA pools of each strain. Each RNA pool was used to generate a sequencing librarie, and was regarded as a biological sample in the present paper. 3 μg total RNA per biological sample was used as the input material for RNA sample preparation. After RNA sample preparation, sequencing libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, E7530) following the manufacturer’s recommendations. In general, mRNA was enriched by oligo(dT) beads. Then the enriched mRNA was fragmented using fragmentation buffer and reverse transcripted into first strand cDNA with random primers. Second strand cDNA was synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit, end repaired, poly(A) added, and ligated to illumine sequencing adapers. Illumine RNA-seq was carried out by Gene Denovo Bitochenology Co. (Guangzhou, China) with an Illumina Hiseq 4000 platform. <b>De novo assembly of reference sequences</b> All raw sequencing reads were trimmed by removing reads containing adapters, reads containing more than 10% of unknown nucleotides, and low quality reads containing more than 50% of low quality bases (Q-value&lt;10). The Trinity software was used to assemble the clean reads of 9 libraries using default parameters (Grabherr et al 2011). The assembled sequences were then filtered using the CD-hit program to reduce redundancy (Li and Godzik, 2006). Sequences shorter than 200 bp were discarded. The resulting sequences, which are called unigenes, were considered as the final non-redundant transcripts and were used as the reference sequences for subsequent analysis. <b>Transcriptome annotation and ontology</b> The unigenes were used as query sequences to search against the NCBI non-redundant (NR) protein database (http://www.ncbi.nlm.nih.gov), the Swiss-Prot database (http://www.expasy.ch/sprot), Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg) and Cluster of Orthologous Group. Searches were conducted using the BlastX program with an E-value cutoff of 1e<sup>-5</sup>. Gene ontology (GO) was performed by importing the NCBI NR Blast results into Blast2GO software. GO terms were then assigned to each sequence automatically. The annotation output was categorized by cellular component, molecular function and biological process. <b>Differential expression gene analysis</b> Clean reads from each RNA sample were aligned to the reference transcriptome using short reads alignment tool Bowtie2 by default parameters (Li et al., 2009). The gene abundances were calculated and normalized to RPKM (reads per kb per million reads). The expression level of each transcript in each sample was then normalized using edgeR. Transcripts with fold change values larger than 2 and p values lower than 0.05, were included in subsequent analyses as the differentially expressed genes (DEGs) (Robinson et al., 2010). <b>GO and KEGG enrichment analysis of DEGs</b> All transcripts were mapped to GO terms in the Gene Ontology database (http://www.geneontology.org/ ) and KEGG database (http://www.genome.jp/kegg/). The GO analysis of transcripts and highly expressed genes was plotted using the web-based program WEGO program (http://wego.genomics.org.cn/ cgi-bin/wego/index.pl) with default parameters. GO terms with corrected P-values less than 0.05 were considered significantly enriched. KOBAS software was used to assess the statistically significant enrichment of DEGs in the KEGG pathway.