Global gene expression analysis data of chicken dendritic cells infected with H9N2 avian influenza virus

This data article reports the global gene expression analysis data of chicken DCs infected with H9N2 avian influenza virus (AIV) compared with mock infection. The differentially expressed genes (DEGs), and the data of GO enrichment analysis and KEGG pathway analysis for DEGs were reported here. In addition, some of these DEGs associated with innate immune response and antigen presentation were also verified by qPCR. The replication of H9N2 AIV in DCs, and the viability kinetic of DCs during H9N2 AIV infection, and the primers for qPCR were also reported in this data article. The data presented here was used on the research article entitled “Transcriptomic profile of chicken bone marrow-derive dendritic cells in response to H9N2 avianinfluenza A virus”.


Specifications table
Immunology and Microbiology Specific subject area The virus infection influence on the gene expression of immune calls. Type of data

Value of the Data
• The first global gene expression analysis of chicken DCs infected with H9N2 AIV.
• These data will help to understand the host immune response to H9N2 infection in chickens.
• Expression analysis data in chicken DCs may be further used for comparative analysis with expression assays in other poultry.

Data description
Here we report the global gene expression analysis data of chicken DCs infected with H9N2 AIV compared with mock infection. The sequence database was deposited to NCBI, and the GEO accession numbers is GSE117163. The data show that 4151 genes were significantly up-regulated, and 2138 genes were significantly down-regulated following H9N2 AIV infection (Supplementary Table 1). GO enrichment analysis of these differentially expressed genes (DEGs) showed that a total of 130 and 120 GO terms were significantly enriched for the up-and down-regulated DEGs respectively, in three main GO categories: cellular components, molecular functions, and biological processes ( Table 1 , Supplementary Tables 2-4). Pathway analysis of the up-regulated and down-regulated DEGs was also performed on the KEGG database ( Table 2 , Supplementary  Table 5). In addition, the phenotype identification of DCs, and the viability kinetic of DCs during H9N2 AIV infection, and the replication of H9N2 AIV in DCs and some of these DEGs were also determined by flow cytometric analysis and qPCR ( Figs. 1 and 2 ), and the primers for qPCR were listed in Table 3 .

Cell culture and virus infection
The bone marrow (BM) monocytes were collected from femurs of four 4-week-old specific pathogen-free (SPF) white leghorn chickens, and were cultured for dendritic cells (BM-DCs) as previously described, with some modifications [2] . Briefly, BM cells were cultured in 6-well plates at a concentration of 5 × 10 6 /ml in RPMI-1640 (Wisent) complete medium containing 5% FBS (Wisent), 100 U/ml penicillin and 100 μg/ml streptomycin for 6 h at 41 °C in 5% CO 2 , and then non-adherent cells were removed by replacing with fresh complete medium containing 50 ng/ml chicken GM-CSF (Abcam, USA), and 10 ng/ml IL-4 (Kingfisher, USA). Half of the medium was replaced with fresh complete medium containing GM-CSF and IL-4 at day 2, 4 and 6. At day 7,   the surface markers of BM-DCs were analyzed by flow cytometry with antibodies as previous experiments [3] , and then BM-DCs were used for the infection of H9N2 AIV. Three H9N2 subtype avian influenza viruses, A/duck/Nanjing/0 6/2003(NJ0 6), A/chicken/Changzhou/0504/2017(CZ0504), and A/chicken/Anhui/S02/2013(AHS02), were propagated in SPF white leghorn chicken eggs respectively, and the allantoic fluid was concentrated via sucrose gradient ultracentrifugation and resuspended in RPMI. The allantoic fluid from mock infected eggs was processed in the same manner and used for mock infection. Viral titers were measured by calculating the 50% tissue culture infectious dose (TCID 50 ) in MDCK cells. Unless otherwise stated in the text, H9N2 AIV refers to NJ06. BM-DCs (2 × 10 6 /ml) were infected with H9N2 AIV (10 6 TCID 50 /0.1 ml) and then were collected for RNA sequencing at 6 h post infection. Three independent biological replicates of the cell culture and virus infection experiments were performed for the RNA sequencing analysis.

RNA sequencing
Total RNA was extracted from BM-DCs using TRIzol reagent (Invitrogen). The integrity and concentration of the extracted RNA were assessed by Agilent 2200 Bioanalyzer (Agilent Table 3 Primers used for qPCR.

Data analysis
The raw reads were filtered by removing the adaptor sequences and low-quality reads containing more than 5% ambiguous bases (noted as N) or more than 20% of bases with qualities of < 20 to obtain clean reads. Thereafter, the clean reads were mapped to the Chicken genome (Version: Gallus_gallus-5.0 NCBI), using HISAT2 with default parameter. The gene expression data were generated and normalized by fragments per kilobase of transcript per million uniquely mapped reads (FPKM) [4] . Differentially expressed genes (DEGs) analysis was performed using DEGSeq algorithm, and DEGs with a p-value < 0.05, a FDR < 0.05 and a fold change > 2 were selected for GO and KEGG pathway enrichment analyses, respectively. The GO and KEGG pathways were considered significantly enriched when FDR < 0.05.

Quantitative real-time PCR
The DEGs recognized by RNA-seq was verified by Quantitative real-time PCR (qPCR). Total RNA was isolated from a replica RNA sequencing infection experiment using TRIzol reagent (Life Technologies) and treated with DNase I (Fermentas, Glen Burnie, MD, USA). One microgram of total RNA per sample was reverse transcribed into cDNA using a PrimeScript RT Reagent Kit (Takara). The qPCR was performed using Talent qPCR PreMix SYBR Green (Tiangen, China) on a Real-Time System Lite TP700 (Takara, Japan). The product specificity of qPCR was verified by one cycle for melting curve analysis. The expression of each cytokine gene relative to that of the β-actin was calculated using the 2 − CT method. All primers for these target genes are listed in Table 3 .