Next-generation sequencing (NGS)

Total RNA samples for sequencing were purified from 20 mg of tissue sample collected from 8 chickens (4 chickens in the CON group and 4 in the GEN treatment group) using the RNeasy Fibrous Tissue Mini messenger RNA (mRNA) extraction kit (Qiagen, Hilden, Germany) following the manufacturer’s recommendations. The concentration and purity of total RNA were checked using a UV/Vis spectrophotometer (ACTGene, New Jersey, USA) at 260 nm, and sample integrity was evaluated using a microfluidic assay on a Bioanalyzer system (Agilent Technologies, Inc., Santa Clara, CA, USA). Only high-quality RNA extracts (RNA integrity number (RIN) ≥8) were used for pooling within each treatment group using equal amounts of RNA per chicken. NGS data were obtained from pooled RNA samples within each group to ensure the most robust transcriptome. Construction of complementary DNA (cDNA) libraries for RNA sequencing was performed using a TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA). RNA-Seq analysis was performed for identification of transcriptional changes using a MiSeq instrument (Illumina) according to the manufacturer’s recommendations (CapitalBio, http://cn.capitalbio.com/) using paired-end libraries⁶². Two replicates of each pool were analyzed independent for library synthesis and sequencing. The quality of the raw reads was assessed using FastQC (Version 0.10.1)⁶³. Adapters, low-quality reads at the 3′end, reads with fuzzy N bases, rRNA, sequences shorter than 20 nt and low-quality reads (those with a Q < 20) were trimmed with the FASTX clipper (Version 0.0.13). All 126-bp double-end reads of 12 samples from both treatment groups were separately aligned to the chicken reference genome (Gallus gallus 5.0, version 81, Ensembl) using the spliced mapping algorithm in TopHat2 (version: 2.0.9)⁶⁴. Unless otherwise stated above, all programs were run with the default parameters. The number of reads equivalent to mapped reads (reads per kilobase per million (RPKM)) was used to normalize the expression of each gene. The quality of obtained data was checked based on the presence and abundance of contaminating sequences, average read length, and GC content. The NGS experiment conformed to the MIAME guidelines⁶⁵, except for the microarray design.