A place supplied grass carp which weighs around 100-250 grams were situated in city of Guangzhou, province of Guangdong, China. These were kept at the temperature of 25-26 °C in a continuous flow water network for around 15 days so as they could acclimate to the laboratory conditions before the experiments were carried out.
Isolation and purification of RBCs
RBCs of grass carp were obtained from the peripheral blood as explained already with minor changes . Briefly, heparinized syringes were used to draw peripheral blood from the caudal vein which is combined with 0.7% buffer saline solution (0.7 g NaCl in 100 ml H2O) containing heparin sodium (0.1 mg/ml). The purification of red blood cells was done by two consecutive density-gradient centrifugations in 34% and 51% Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden) at 500 g for 35 minutes at 4°C. The RBCs were gathered and washed three times in a 0.7% buffer saline solution, and then resuspended in an L-15 medium (Invitrogen, USA) supplemented with 100 µg/ml streptomycin (Sigma, USA) and, 10% fetal bovine serum (FBS), 100 U/ml penicillin, and at a density of 106 cells/ml at 28 °C. All grass carp tests were done in careful observance with the guidance of the Ministry of Science and Technology of China 2006 and affirmed by the Zhongkai University of Agriculture and Engineering’s Animal Ethics Committee.
Infection of RBCs with A. hydrophila
A.hydrophila was maintained and cultured according to our routine laboratory protocols . Subsequently, the experimental RBCs (1 × 106 cells/ml) from 6 fish were divided into control and the infected groups. The RBCs in the infected group were incubated at a 1:10 ratio with A. hydrophila for 12 hours under constant rotation at 28°C, whereas the RBCs in the control group were incubated with the similar amount of PBS. After 12 hr treatment, three equal specimens were combined as biological replicas.
Extraction, RNA-Seq library construction, and sequencing of RNA
Complete RNAs were removed from the A. hydrophila-infected and non-infected RBCs using RNAiso plus made in Takara, Japan as indicated by the maker’s procedure. Subsequently, the characteristics and amount of the complete RNA were identified utilizing Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and Nano Drop 2000 (Thermo Scientific), respectively. The libraries were built using the TruSeq™ RNA Sample Prep Kit (Illumina, San Diego, CA, USA) according to the maker’s directions. Afterward, the three RNA-Seq libraries were carried out with 150/125 bp paired-end sequencing on the BGISEQ-500 platform (BGI, Shenzhen, China) as per the last record .
Sequence filtering, gene annotation, and de novo assembly
The transcriptomic test was performed by the strategy described before head . The generated raw sequencing readings were firstly clean up by eliminating adaptor sequences and reduced quality sequences (over 20% of the basic qualities were below than 10) using the tool FASTX, which is utilized to decrease data clamor and clean raw readings. The informational collection of the source of rRNA arrangements was acquired from general rRNA databases. The remaining clean readings were planned to a C. idellus reference genome utilizing HISAT. After mapping of genome, StringTie were utilized to rebuild transcripts, and with genome annotation information, the coding capability of those latest transcripts predicted utilizing CPC and the novel transcripts were identified using the Cuffcompare tool. Gene expression abundance was evaluated utilizing the RPKM (readings-per kilobase per million mapped reads) method. All reference genes were performed with a sequence based on the NCBI nr (non-redundant) protein with an E-value threshold of 10−5 and the Uniprot database. Subsequently, all differential expressed genes (DEGs) were mapped to the GO terms data for functional annotation, the KOG, KEGG for pathway-enrichment analysis.
DEGs, KEGG, and GO enrichment analysis
Based on the obtained levels of gene expression, DEGs between A. hydrophila non-infected and infected groups were utilized with criteria with a FC (fold change) of gene expression > 2.0 (complete value of the log2 Ratio ≥ 1) and FDR (False Discovery Rate) ≤ 0.001. To know more about the DEGs and pathways responding to the infection of A. hydrophila, functional groups and pathways encompassing the DEGs were plot to terms in the KEGG and GO databases with the Blast2 GO pipeline and KEGG enrichment analysis utilizing KOBAS software, respectively.
- hydrophila infection and immune genes expression
Six cytokines genes (CCL4, CCL11, CCL20, IL4, IL12, and IFNα) were analyzed by qRT-PCR to further validate RNA-seq data. The reverse transcription reaction was in a total volume of 20 μl, which contains 10 μl AceQ® qPCR SYBR® Green Master Mix (Vazyme, Nanjing, China), 1 μl cDNA, 7 μl DEPC treated water, and 1 μl of each particular primer (list in Table 1). The quantitative real-time PCR cycling conditions were as stated: 95 °C for 3 minutes, further at 95 °C for 15 seconds of 40 cycles, for 30 seconds at 60 °C, for 20 seconds at 72 °C, and finally for 5 minutes at 4 °C . All reactions of each gene in each sample were carried out in triplicate. Meanwhile, an analysis of melting curve was performed to identify the nature of target. The relative expression ratio of the β-actin gene versus the target genes was calculated using the 2-∆∆CT method , and all the information was provided in terms of relative mRNA expression.
Examination of apoptosis in A. hydrophila-infected RBCs
The RBCs (1 × 106 cells/ml) were separated into two different groups; infected and control. The RBCs in the infected group were incubated with A. hydrophila at a ratio of 1:10 for 6, 12, and 24 h under constant rotation at 28 °C, respectively. The control group was incubated with the equal proportion of PBS. After 6, 12, and 24 h of infection, a kit called the Alexa Fluor® 488 annexin V/Dead Cell Apoptosis Kit (Invitrogen, USA) was utilized to test the apoptosis of RBCs as stated earlier . After staining, the RBCs were viewed by fluorescence microscopy (Leica DMI8, Germany). Furthermore, to additionally explain the mechanism of apoptosis, some apoptosis pathway-related genes; including p53, Fas, LRDD, and caspase 8 were determined by qRT-PCR. All primers were shown in Table 1.
Statistical evaluation was carried out by utilizing SPSS software version 17.0. Whole formation was depicted as a mean ± SD (standard deviation) at least 3 separate tests. The statistical importance of the information was evaluated by ANOVA (one-way analysis of variance), and GraphPad Prism 7 was used to draw figures . The difference was considered important when P < 0.05 (*).