2.5. Differential expression analysis via Illumina cDNA library sequencing
To prepare the Illumina library, total RNA was extracted from brain and ganglia tissues of three individuals as described above, and the mRNAs were enriched with Oligo (dT) mRNA magnetic beads following the manufacturer’s instructions. The cDNA libraries were synthesized using mRNA fragments as templates. The quality of four cDNA libraries were checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and 150 bp paired-end reads were generated by Illumina HiSeq 2000 platform. The unigene sequences generated by SMART sequencing were used as a reference, and the clean reads obtained from Illumina sequencing were aligned to the reference using bowtie2 (Langmead & Salzberg 2012), to analyze gene expression counts. The fragments per kilobase of transcript per million mapped reads (FPKM) value for the gene expression level were calculated using eXpress (Roberts & Pachter 2013). Based on gene expression counts obtained from each sample, differentially expressed genes were identified using DESeq (Anders S 2012). The thresholds for significant differential expression were set as a p-value < 0.05 and absolute of log2(fold change) >2. Significance was tested using hypergeometric distribution. Finally, differentially expressed genes were used for GO and KEGG enrichment analysis.