RNA Sequencing and Bioinformatic Analysis

RNA sequencing and bioinformatics analysis was conducted in collaboration with the Mayo Medical Genomics Facility as previously described [12 (link)]. Quality of the RNA was assessed by the Mayo Gene Expression Core using Agilent Bioanalyzers. All samples had RNA Integrity Numbers greater than 7.0. RNA sequencing was performed as paired-end base reads on an Illumina HiSeq 2000 with three samples per lane, using the TruSeq SBS Sequencing Kit, Version 3. Base calling was performed using Illumina’s RTA version 1.12.4.2. Bioinformatics were performed with the assistance of the Mayo Division of Biostatistics and Informatics. Analysis of each sample (alignment statistics, in-depth quality control metrics, and gene and exon expression levels) was done using Mayo Clinic’s MAPRSeq v1.2. Reads were mapped using Tophat version 2.0.6 against the hg19 reference genome and gene counts were produced using htseq. Differential expression analyses between samples were computed using an edgeR version 3.3.8 algorithm. Data was deposited in the NCBI Gene Expression Omnibus (accession number GSE86007).