RNA-Seq of SW480 Control 3
RNA sequencing libraries were prepared using the TruSeq RNA library prep kit v2 (Illumina) following the manufacturers recommended protocol. The libraries were sequenced using single-end 50-cycle reads on a HiSeq 2500 sequencer (Illumina) at the University of Michigan DNA Sequencing Core Facility. Raw sequencing read quality was assessed utilizing FastQC. Reads were aligned to the reference human transcriptome (hg38) using Bowtie v 2.1.0.0 and TopHat v2.0.9. Default parameters were used for the alignment, with the exception of -b2-very-sensitive, -no-coverage-search, and -no-novel-juncs. Mate inner pair distances were estimated by TopHat, and the values were used in the alignment. Expression quantification and differential expression analysis between HIF-31-expressing and parental HT29 or SW480 cells were conducted using CuffDiff v 2.1.1 with the parameter settings -multi-read-correct, -compatible-hits-norm, and -upper-quartile-norm for normalization of expression calculations across samples. For the CuffDiff analysis, we used hg38.fa as the reference genome and hg38.gtf as the reference transcriptome. Genes were considered differentially expressed between conditions at a false-discovery rate-adjusted p value of less than 0.05.