Profiling gene expression changes in primary ovarian tumors compared to matched normal fallopian tubes

Ovarian cancer is the deadliest gynecologic malignancy and the fifth leading cause for cancer related deaths among women in USA. For a comprehensive understanding of the gene expression changes accompanying ovarian carcinogenesis, we isolated RNA from 8 pairs of matched FT and primary tumors from OC patients and sequenced them. Library preparation and next generation RNA sequencing was carried out at the Center for Genomics and Bioinformatics core facility, Indiana University, Bloomington. The library preparation was done using TruSeq Stranded mRNA HT Sample Prep kit (Illumina cat#RS-122-2103) according to the manufacturer’s protocol and 8-neucleotide barcodes were added for multiplexing. The barcoded libraries were cleaned by bead cut with AMPure XP beads (Beckman Coulter, cat#A63882), verified using Qubit3 fluorometer (ThermoFisher Scientific) and 2200 TapeStation bioanalyzer (Agilent Technologies), and then pooled. The pool was sequenced on NextSeq 500 (Illumina) with NextSeq75 High Output v2 kit (Illumina cat#FC-404-2005). 8 pairs of matched primary ovarian tumors and fallopian tubes from ovarian cancer patients were used for sequencing. The library preparation was done using TruSeq Stranded mRNA HT Sample Prep kit (Illumina cat#RS-122-2103) according to the manufacturer’s protocol and 8-neucleotide barcodes were added for multiplexing. The barcoded libraries were cleaned by bead cut with AMPure XP beads (Beckman Coulter, cat#A63882), verified using Qubit3 fluorometer (ThermoFisher Scientific) and 2200 TapeStation bioanalyzer (Agilent Technologies), and then pooled. The pool was sequenced on NextSeq 500 (Illumina) with NextSeq75 High Output v2 kit (Illumina cat#FC-404-2005). Reads were adapter trimmed and quality filtered using Trimmomatic ver. 0.33, with the cutoff threshold for average base quality score set at 20 over a sliding window of 3 bases. Reads shorter than 20 bases post-trimming were excluded. Cleaned reads mapped to human genome GRCh38 with gencode v25 annotation using Tophat2 ver 2.1.0. Significantly differentially expressed genes at 5% FDR with at least 2 fold change were called using DESeq2 ver. 1.12.3.