Genome-wide transcriptome analysis by RNA sequencing (RNA-Seq)

Total RNA from mouse skin was used to create ribosomal RNA-depleted cDNA libraries (NEBNext® Ultra^™ RNA Library Prep Kit). Each sample was sequenced to 40 million raw reads in a single end 75bp sequencing run on the Illumina NextSeq500, demultiplexed and converted to FastQ data. The NIAMS Biodata Mining and Discovery Section mapped FastQ data to the mouse genome build mm10 using TopHat 2.1.1. Gene expression values (RPKM, reads per kilobase exon per million mapped reads) were calculated and log2 transformed (with a 0.1 offset). Differentially expressed genes (DEG) were determined by ANOVA, and a DEG heat map was created with Partek Genomic Suite. DEGs were defined as having FDR (q-value) ≤ 0.05 and with raw FC ≥ 1.8. Ingenuity Pathway Analysis (Qiagen: www.ingenuity.com) used Fishers exact test to detect significantly enriched (P-value < 0.05) upstream regulators and disease pathways from expression data.