Total RNA was isolated from human pancreatic cancer cell lines as previously described. Sequencing libraries were prepared from 100–500 ng of total RNA using the TruSeq RNA sample preparation kit v2 (Illumina, San Diego, CA) according to the manufacturer's protocol. Briefly, mRNA was purified, fragmented and used for first- and second-strand cDNA synthesis followed by adenylation of 3′ ends. Samples were ligated to unique adaptors and subjected to PCR amplification. Libraries were validated using the 2100 BioAnalyzer (Agilent, Santa Clara, CA), normalized and pooled for sequencing. RNA-seq libraries from three biological replicates for each experimental condition were sequenced on the Illumina HiSeq 2000 using barcoded multiplexing and a 100-bp read length. Image analysis and base calling were done with Illumina CASAVA-1.8.2. This yielded a median of 12.4 million usable reads per sample. Short read sequences were mapped to a UCSC hg19 reference sequence using the RNA-seq aligner STAR. Known splice junctions from hg19 were supplied to the aligner and de novo junction discovery was also permitted. Differential gene expression analysis, statistical testing and annotation were performed using Cuffdiff 2. Transcript expression was calculated as gene-level relative abundance in fragments per kilobase of exon model per million mapped fragments and employed correction for transcript abundance bias. RNA-seq data is deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA accession number SRP057571). Functional enrichment analysis was limited to gene ontology terms and examined using default parameters in the web-based Database for Annotation, Visualization and Integrated Discovery (DAVID, v6.7; http://david.abcc.ncifcrf.gov) Full paper
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