Pathways analysis

Gene expression data obtained previously^15,16, was evaluated for Ingenuity Pathway Analysis (IPA) (Ingenuity System Inc., Redwood City, CA). Briefly, human corneal epithelial cells (HCEC) were cultured on silk films with flat, 2000, 1000, and 800 nm ridge width topography. After 72 h in culture, cells were collected and total RNA obtained and used to construct a cDNA library by using TruSeq mRNA-seq Sample Preparation Kit (Illumina, San Diego, CA, USA). Illumina Hiseq 2500 sequencing system (Illumina, Inc., San Diego, CA, USA) was used for sequencing. Alignment of RNA-Seq reads against the human reference genome, were performed on SeqMan NGen assembler (DNAstar version 2016, Madison, WI, USA). The assembly was analyzed by DNAstar Qseq to generate normalized expression values of the transcript isoforms. Fold change between samples collected from different experimental conditions and the P value for each transcript were analyzed by ANOVA. Data set containing differentially expressed gene identifier and corresponding expression values were uploaded to Ingenuity Pathways Analysis. IPA uses the global molecular network developed in Ingenuity Pathways Knowledge Base (IPKB) which contains the biological function, interaction, and related biochemical pathways.