NextSeq 500 sequencing; The intercellular signaling network of ovarian cancer metastases
The intercellular signaling network of ovarian cancer metastases
Ascites or omentum was collected from patients with high-grade serous ovarian carcinoma undergoing primary surgery at the University Hospital in Marburg. Informed consent was obtained from all patients according to the protocols approved by the local ethical committee  Metastasized omentum was digested with Trypsin for 30 min. Isolated cells were cryopreserved. Tumor cells were isolated from Cryo using a CD45/EpCAM-MACS depletion and dissolved in TriFast. Purity was determined via flow cytometry. Cells were extracted ex-vivo and sequenced without culture. RNA-extraction with TriFast (1ml) according the manufacturer protocol. RNA is dissolved in RNase-free water (with RNase-Inhibitor Superase-IN 1:20). DNase digestion with DNA-freeâ„¢ Kit (invitrogen, ThermoFisher scientific) followed. The integrity and purity of RNA was determined using the Experion RNA StdSens  Illumina TruSeq stranded mRNA according to manufactures' instructions