RNA-seq analysis
Total RNA was extracted from the back skin lesions of the IMQ-treated
and high-dose allicin-treated groups for transcriptome sequencing. RNA
was isolated and purified using Trizol reagent (Invitrogen, Carlsbad,
CA, USA). The RNA amount and purity of each sample were quantified using
NanoDrop ND-1000 (Wilmington, DE, USA). The final cDNA was sent for
library preparation and sequencing on an Illumina Novaseq™ 6000 at LC
Bio Technology (Hangzhou, China). With StringTie and Ballgown, we
estimate the expression levels for all transcripts and calculate the
expression levels for mRNAs with FPKM (FPKM = [total exon
fragments/mapped reads (millions) × exon length (kB)]). The
differentially expressed genes (DEGs) were selected by R package edgeR
or DESeq2 with fold change > 2 or fold change <
0.5 and p-value < 0.05. Following that, we conducted gene
ontology (GO) enrichment and Kyoto encyclopedia of genes and genomes
(KEGG) enrichment analyses of the differentially expressed mRNAs in the
two groups.