qRT-PCR Validation of RNA-Seq Results

To corroborate the RNA-Seq results, we randomly selected 10 genes from the SDEGs for qRT-PCR validation, including five upregulated genes and five down-regulated genes. Chicken spleen tissue total RNA was extracted, and a reverse transcription reaction was carried out using a primerScriptTM RT kit (Takara, Kyoto, Japan) to obtain cDNA for qRT-PCR. The reaction was carried out using the LightCycler^® 96 instrument qRT-PCR system (Roche, Basel, Switzerland) and SYBR^® PremixEx TaqTM kit (Takara, Kyoto, Japan). The qRT-PCR amplification procedure was as follows—95 °C for 3 min, 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and extended at 72 °C for 10 min. In addition, before the gene quantification, three common chicken housekeeping genes (B2M, β-actin and GAPDH) were tested [16 (link)] and evaluated using GeNorm [17 (link)]. Since GAPDH showed a lower M value and was stable in Gushi chickens [18 (link)], it was used as an internal reference gene. The relative expression changes of the genes were calculated using the 2^−ΔΔCt method. The qRT-PCR samples were the same as the RNA-seq samples, with three biological repeats for each group and three repetitions for each sample. The primer sequences are listed in (Additional File 3: Table S2).