Profiling gene expression changes in ovarian cancer cells seeded on 3D organotypic culture of omentum

Ovarian cancer is the deadliest gynecologic malignancy and the fifth leading cause for cancer related deaths among women in USA. Most patients have metastatic disease at the time of diagnosis and this causes the poor prognosis. Regulation of early steps of metastatic colonization - the rate limiting step of metastasis - is poorly understood. Using an organotypic 3D culture model of the omentum, we have mimicked the early stages of metastatic colonization in vitro. Using this, we have profiled the mRNA expression changes occurring in high grade serous ovarian cancer cell line Kuramochi, OVCAR4 and OVCAR8 when they are seeded on the 3D omentum culture compared to controls seeded on normal culture dishes. The ovarian cancer cells were separated by FACS after 2 days of culture and their RNA was isolated and RNA-seq was performed. 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). Overall design: Kuramochi, OVCAR4 and OVCAR8 cells seeded on the 3D omentum culture or on plastic culture dishes (n=3/cell line) 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.