title: Illumina Genome Analyzer II sequencing; Deregulation of PPAR-beta/delta target genes in tumor-associated macrophages by fatty acid ligands in the ovarian cancer microenvironment - RNAseq
design: Deregulation of PPAR-beta/delta target genes in tumor-associated macrophages by fatty acid ligands in the ovarian cancer microenvironment - RNAseq
library_construction_protocol: Ascites was collected from 30 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 committe Cells were isolated on June 06th 2013 from healthy human donor blood of unknown gender via LSM1077 gradient centrifugation. Adherent cells were positively selected on primaria 6 well dishes (3x10^6 monocytes per well in 2 ml PBS (PAA)) followed by 6 washing steps with PBS. Finally the monocytes were cultured for 6d in humidified incubator at 37  degrees C, 5 % CO2 in 2ml RPMI medium supplemented with 10 % FCS (both PAA). Cells were treated with control (DMSO), L165 1 uM final concentration or ST247 300 nM final concentration for 1d. Cells were washed with PBS, supernatant was removed completly, then 1 ml of peqGOLD TriFast (PEQLAB) was added to each well and transfered to a 1,5 ml Eppi. Following 5 min of incubation at RT 200 ul trichloromethane were added to the solution. The probes were mixed thoroughly every 3 minutes during a 10 minute incubation at RT. Probes were centrifuged at 13.000 g for 15 min at 4  degrees C then the aquouse top phase was transfered to a new eppi containing 600 ul of ice cold 2-propanol. Probes were mixed and centrifuged for 10 min at 13.000 g, 4  degrees C. The supernatant was removed and the pelleted RNA was washed twice with 75 % Ethanol for 5 minutes at 13.000 g, 4  degrees C. The pellet was dried and dissolved in 15 ul of RNase free water following quality control using Xcalibur (Thermo scientific). Libraries were created using the Illumina Truseq mRNA kit used according to the manufacturer's instructions.
identifiers.namespace: INSTITUTE FOR MOLECULAR AND TUMOR BIOLOGY PHILIPPS UNIVERSITY  MARBURG GERMANY
identifiers.id: E-MTAB-3167:ST003
identifiers.namespace: Institute for molecular and tumor biology Philipps University  Marburg Germany
identifiers.id: E-MTAB-3167:ST003