Illumina HiSeq 2000 paired end sequencing; RNA-seq of isolated tumor cells from high grade serous ovarian cancer (HGSOC) patients
RNA-seq of isolated tumor cells from high grade serous ovarian cancer (HGSOC) patients
Tumor cell isolation. Ascites: Fresh ascites was filtered consecutively for tumor cell aggregates (spheroids) using CellTrics® filters with mesh sizes of 150 μm, 30 μm, and 20 μm. Spheroids were isolated from the washed 30 μm-filter and single cells were isolated from the 20 μm filtrate. For gene expression analysis, EpCAM+ cells were isolated from the 20 μm filtrate with Dynabeads Epithelial Enrich (Invitrogen, Carlsbad, CA, USA) and a magnetic bead-based cell separation device. The ascites EpCAM+ single cells and the ascites spheroids were lysed with QIAzol and stored at −80°C for subsequent RNA preparation. Tumor tissue: Tissue slices of ovarian and peritoneal tumor masses were obtained during surgery, minced, and digested with 1.04 U/ml Liberase DH research grade (Roche, Basel, Switzerland) while stirring at 37°C for one hour. A representative piece of this tissue was fixed, paraffin embedded, and a hematoxylin-eosin stained section was examined by the pathologist. The cell suspension was supplemented with 4 mM EDTA and depleted of undigested tissues using a 40 μm cell strainer. The filtrate was used for enrichment of EpCAM+ cells as described above. Total RNA isolation (>200 nt): Total RNA from QIAzol lysed cell samples was isolated using the miRNeasy® Mini Kit (Qiagen, Hilden, Germany) and concentration quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA) and RiboGreen RNA Reagent (Invitrogen, Carlsbad, CA, USA). Quality was assessed by an Agilent 2100 Bioanalyzer (Santa Clara, CA, USA) and only samples with an RNA Integrity Number (RIN) > 7.7 were used for library preparation. NEXTflex RNA-Seq: Sequencing libraries were prepared from 200 ng total RNA isolated with the miRNeasy Mini Kit (Qiagen, Hilden, Germany). RNA quality was controlled on a RNA Pico Chip (Bioanalyzer 2100, Agilent, Santa Clara, CA, USA) and quantified with NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA) and RiboGreen RNA Reagent (Invitrogen, Carlsbad, CA, USA). Ribosomal RNAs were removed using the Ribo-Zero™ rRNA Removal Kit (Epicentre, Madison, WI, USA) and libraries prepared employing the NEXTflex RNA-Seq and Barcoding Kits (Bioo Scientific, Austin, TX, USA) with halved reaction volumes with an epMotion 5075 automated pipetting system (Eppendorf, Hamburg, Germany) according to the manufactures instructions. Quality of libraries was assessed on a DNA High Sensitivity chip (Bioanalyzer 2100) and quantified with a digital PCR system (QX100™ Droplet Digital™ PCR System, BioRad, Hercules, CA, USA) and the ddPC Library Quantification Kit (BioRad). Eight libraries were pooled equimolarly.