title: NextSeq 500 sequencing; The intercellular signaling network of ovarian cancer metastases
design: The intercellular signaling network of ovarian cancer metastases
library_construction_protocol: 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  Tumor-free\" omental tissue was digested with Collagenase (370U/ml), freshly added to adipocyte digestion buffer (5mM D-Glucose, 1.5% BSA in PBS)for 1h at 37°C (rotating). After centrifugation and washing (with PBS), 250 μl adipocytes were dissolved in 2 ml TriFast (ex vivo). Purity was tested via immunofluorescence. No EpCAM+ or CD45+ cells were visible. 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-free™ 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