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.  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.  Protein A sepharose beads (GE Healthcare) were washed twice 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) und blocked overnight  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  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). 300 µl of a  15 min 20,000xg supernatant was used for overnight IP after preclearing with 50 µl of protein A sepharose coupled to 10 µg of rabbit preimmune IgG for 45 min at 4 ˚C.  On the next day, immunoprecipitation was performed with 50 µl of blocked protein A sepharose slurry. Beads were washed at 4 ˚C once each with 1 ml of wash buffers I (20 mM Tris pH 8,1; 150 mM NaCl; 1% (v/v) Triton X-100; 0,1% (w/v) SDS; 2 mM EDTA) and II (20 mM Tris pH 8,1; 500 mM NaCl; 1% (v/v) Triton X-100; 0,1% (w/v) SDS; 2 mM EDTA) followed by two washes with wash buffer III (10 mM Tris pH 8,1; 250 mM LiCl; 1 % (v/v) NP40; 1 % (w/v) sodium deoxycholate; 1 mM EDTA). Beads were washed twice with Qiagen buffer EB at room temperature. DNA complexes were eluted twice each with 200 µl of 1% (w/v) SDS and 100 mM NaHCO3 for 15 min with agitation. Reversion 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.  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.