title: Illumina HiSeq 1500 sequencing; ChIP-seq of tumour associated macrophages derived from high grade ovarian serous carcinoma patient ascites
design: ChIP-seq of tumour associated macrophages derived from high grade ovarian serous carcinoma patient ascites
library_construction_protocol: Ascites were 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.  Cells were purified from ascites in a Ficoll gradient. 1 h adherence selection in Greiner flasks (no. 660175) in autologous ascites. Fixation was done using 1% formaldehyde for 10 min at room temperature followed by quenching with 125 mM glycin for 5 min. Washed with PBS twice. Lysis in hypotonic lysis buffer (5 mM PIPES pH 8,0; 85 mM KCl; 0,5 % (v/v) NP40) for 20 min on ice. Nuclei were resuspended in ChIP RIPA (10 mM Tris-HCl pH 7,5; 150 mM NaCl; 1 % (v/v) NP40; 1 % (w/v) sodium deoxycholate; 0,1 % (w/v) SDS; 1 mM EDTA) at 2.5*10^7 cells/ml, incubated for 10 min on ice and sheared in 1 ml aliquots with a Branson S250D using a microtip (52 pulses of 1 s, 2 s pause, 20% amplitude) with coolling (ice-ethanol). Reversion of crosslinks was done overnight at 65 ˚C after adding 10 µg of RNase A and 20 µg of proteinase K in the presence of 180 mM NaCl, 35 mM Tris-HCl pH 6.8 and 9 mM EDTA. Samples were purified using the Qiagen PCR purification kit according to the manufacturer's instructions. 250 ng of DNA were used for MIRA as per the kit's instructions (Methylcollector Ultra, Active Motif, http://www.activemotif.com/catalog/43/methylcollector-ultra).  For details on MIRA, see Rauch et al. 2005 (http://dx.doi.org/doi:10.1038/labinvest.3700311 ),  which describes the use of MBD2 and MBD3 domains as probes for the methylated CpG island recovery assay (MIRA) After-assay DNA was quantified with Picogreen (Life Technologies) on a Nanodrop 3300 Fluorimeter according to the manufacturer's instructions. ChIP-seq libraries were prepared with the Diagenode Microplex kit.