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.