title: RNA-seq of Gallus gallus (SPF Leghorn): Spleen tissue after PBS immunization and NDV challenge, collected 3 days post-challenge
design: This study aims to investigate the differential gene expression in the spleen of SPF Leghorn chickens following immunization and Newcastle disease virus (NDV) challenge, and to elucidate the mechanisms by which in ovo immunization provides long-term protection to the host. The experiment included two groups: one group was immunized with phosphate-buffered saline (PBS), and the other group was immunized with NDV. At 360 days post-hatch (dph), three chickens from each group were randomly sacrificed at 3 days post-challenge (dpc), and their spleens were collected for RNA-Seq analysis. RNA extraction was performed using RNAiso Plus (Takara, Beijing, China), and RNA quality was assessed by agarose gel electrophoresis and a nanophotometer (NanoPhotometer-NP80, implen, Germany). Subsequent RNA extraction, library preparation, and sequencing were conducted at Beijing Biomics Biotech Co., Ltd., using paired-end sequencing on the Illumina Novaseq6000 platform. High-quality sequencing reads were processed and filtered using fastp version 0.18.0, and rRNA-associated reads were removed using Bowtie2 aligner, version 2.2.8. The raw reads were evaluated with FASTQC and NGS QC TOOLKIT, and clean reads were obtained by eliminating reads containing poly-N and low-quality reads. The clean reads were then aligned to the Gallus gallus-5.0 reference genome (GenBank accession number: GCF_000002315.4). Differential expression analysis was carried out using the DESeq R package (version 1.10.1), and differentially expressed genes (DEGs) were identified based on criteria of P < 0.05 and fold change (FC, |log2 FC| 1) with a false discovery rate (FDR < 0.05). The primary biological functions of the DEGs were categorized and delineated through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, with significant GO terms and pathways enriched under a P-value < 0.05. Protein-protein interaction (PPI) network analysis was performed using String v10, and the results were visualized using Cytoscape (v3.7.1) software.