Trueseq mrna sample preparation kit

Total messenger RNA (mRNA) was prepared using Trizol reagent (Thermo–Fisher Scientific) from two experiments, one of which has been previously reported (Phillips et al., 2016 (link)), each with two independent samples. Sequencing libraries were prepared using an Illumina TrueSeq mRNA sample preparation kit. Messenger RNA was purified and converted to double-stranded complementary DNA (cDNA). Adapters and specific indexes were added to each sample. RNA sequencing was performed on a HiSEqn 2500 sequencer analyzer (Illumina, San Diego, CA). Sequencing reads were analyzed using CLCBio Genomic Workbench software (CLC Bio, Aarhus, Denmark) (Wickramasinghe et al., 2014 ). Quality control (QC) analysis was performed using the application NGS quality control tool of CLC Genomics Workbench software (CLC Bio) (Cánovas et al., 2013 (link)). Sequenced single reads (100 bp) were assembled against the annotated human reference genome (release 80) (ftp://ftp.ensembl.org/pub/release-80/genbank/homo_sapiens/). Data were normalized by calculating the ‘reads per kilobase per million mapped reads’ (RPKM) for each gene.

For RNA-seq, RNA quality was determined using Agilent 2100 Bioanalyzer. Samples used for sequencing had RNA integrity numbers (RIN) > 8. Library preparation using Illumina TrueSeq mRNA sample preparation kit was performed at the Weill Cornell Medical College Genomic Core facility, and RNAs were sequenced on Illumina HiSeq 2000 machines. RNA reads were aligned to the mm9 build of the mouse genome using Tophat. Transcript assembly and differential expression was done using Cufflinks with Refseq mRNAs to guide assembly (52 (link)). Analysis of RNA-seq data was performed using the cummeRbund package in R (53 (link)). DAVID bioinformatic database was used to find enriched functional GO-term annotations in RNA-seq data (https://david.ncifcrf.gov/content.jsp?file=citation.htm). Box-and-whisker plots were generated using Prism5 software (GraphPad) to determine the significance between two groups. Error bars plotted on graphs denote SD. Unpaired student’s t test was used to determine the significance between two groups: *P<0.05; **P<0.01; ***P < 0.001; ****P<0.0001; ns, not significant.

FACS isolated keratinocytes were sorted directly into TRI Reagent (Sigma). Three animals were pooled per condition and all experiments were performed in duplicate. RNA was purified using Direct-zol RNA MiniPrep kit (Zymo Research) per manufacturer’s instructions. Quality of the RNA for sequencing was determined using Agilent 2100 Bioanalyzer, all samples used had RNA integrity numbers (RIN) > 8. Library preparation using Illumina TrueSeq mRNA sample preparation kit was performed at the Weill Cornell Medical College Genomic Core facility, and RNAs were sequenced on Illumina HiSeq 2000 machines. Alignment of reads was done using Tophat with the mm9 build of the mouse genome. Transcript assembly and differential expression was determined using Cufflinks with Refseq mRNAs to guide assembly (Trapnell et al., 2010 (link)). Analysis of RNA-seq data was done using the cummeRbund package in R (Trapnell et al., 2012 (link)). Differentially regulated transcripts were used in Gene Set Enrichment Analysis (GSEA) to find enriched functional GO annotations (Subramanian et al., 2005 (link)). MEME software suite (including TomTom) was used to identify enriched motifs in Skint promoters, the JASPAR vertebrate database was as a source for consensus transcription binding site sequences (Bailey et al., 2009 (link)).