title: Illumina HiSeq 1500 sequencing; Deregulation of PPARbeta/delta target genes in tumor-associated macrophages by fatty acid ligands in the ovarian cancer microenvironment -  ChIPseq
design: Deregulation of PPARbeta/delta target genes in tumor-associated macrophages by fatty acid ligands in the ovarian cancer microenvironment -  ChIPseq
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 After Ficoll gradient purification, cells were incubated in Greiner flasks in autologous ascites for 1 h at 37 degrees C in a humidified incubator with 5% CO2 (adherence selection). Nonadherent cells were removed by two PBS washing steps. Using a final concentration of 1% (w/v) formaldehyde for ten minutes at room temperature, proteins and DNA were crosslinked. After adding glycine to a final concentration of 125 mM, the cells were washed twice with ice-cold PBS. After harvesting by scraping and centrifugation, the pellet was resuspended in 1 ml hypotonic ChIP lysis buffer per 8x10^6 cells with protease inhibitors (Sigma) and incubated for 20 minutes. After centrifugation, nuclei were resuspended in ChIP lysis buffer II (RIPA) containing protease inhibitors and sheared with a Branson S250D using a microtip (52 pulses of 1 s, 2 s pause, 20% amplitude) with coolling (ice-ethanol). A 15 min 20,000xg supernatant was used for IP (after preclearing). ChIP was performed as in Adhikary and Mueller, Peroxisome Proliferator-Activated Receptors (PPARs) Methods in Molecular Biology Volume 952, 2013, pp 175-185.  DNA was purified using Qiagen Minelute columns.  Preceding the PE washing step, the membranes were washed twice with pure methanol in order to remove contaminating DNA-binding lipids that inhibit subsequent low-temperature enzymatic modification steps, which we found to be present in samples from primary macrophages. Libraries were synthesized from 1-2 ng of genomic DNA using the MicroPlex kit (Diagenode, Seraing, Belgium).
identifiers.namespace: INSTITUTE FOR MOLECULAR AND TUMOR BIOLOGY PHILIPPS UNIVERSITY  MARBURG GERMANY
identifiers.id: E-MTAB-3166:TA019
identifiers.namespace: Institute for molecular and tumor biology Philipps University  Marburg Germany
identifiers.id: E-MTAB-3166:TA019