Oligo dt directed reverse transcription

For RNA-seq, total RNA was isolated from triplicate sets of human keratinocytes at 48h after transfection using RNAeasy column purification (Qiagen, Germantown, MD). cDNA library was prepared via oligo-dT-directed reverse transcription (Ambion, Grand Island, NY), and subject to deep sequencing on Illumina HiSeq2000 (50bp single-read sequencing) and analyzed at Duke Genome Sequencing & Analysis Resource. The analysis pipeline includes the initial QC to remove sequencing adaptors and low quality bases to facilitate mapping using fastx toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html). High quality reads were mapped to the human reference genome (hg19) using tophat (Trapnell et al., 2009 (link)). The gene differential expression analysis was performed between control and treatment using cuffdiff with upper quartile normalization, multi-hit correction and 0.5% FDR(Trapnell et al., 2013 (link)). Three biological replicates were used for statistical analysis of differential expression for each comparison. Pathway analyses were performed using WEB-based GEne SeT AnaLysis Toolkit (Wang et al., 2013 (link); Zhang et al., 2005a (link)). SYBR green-based real-time RT-PCR was performed in Bio-Rad iCycler with primers listed in (Table S4). Ribosomal 18S RNA or GAPDH was used as an internal control.