Illumina HiSeq 2000 sequencing; ChIP-seq of CSF-1R, H3K4me1, H3K4me3 in human primary unstimulated monocytes and monocytes differentiated 72h with CSF-1 (3 healthy donors buffy coat) and in monocytes from two CMML patients and one healthy donor, EGR1 ChIP-seq in unstimulated monocytes (3 healthy donors buffy coat)
ChIP-seq of CSF-1R, H3K4me1, H3K4me3 in human primary unstimulated monocytes and monocytes differentiated 72h with CSF-1 (3 healthy donors buffy coat) and in monocytes from two CMML patients and one healthy donor, EGR1 ChIP-seq in unstimulated monocytes (3 healthy donors buffy coat)
Peripheral blood mononucleated cells (PBMC) were obtained from healthy donor buffy coats (Etablissement Français du sang, Rungis, France) or CMML whole blood, separated on Pancoll (Pan-Biotech, Dutscher, Brumath, France). Peripheral blood CD14+ monocytes were sorted with magnetic beads and the AutoMacs system (Miltenyi Biotech, Paris, France).  After sorting, monocytes were cultured overnight at 1 million cells/mL in RPMI 1640 Glutamax medium (ThermoFisher Scientific) supplemented with 10% heat inactivated fetal bovine serum (FBS, Lonza, Amboise, France), 1% penicillin/streptomycin and 2mM L-Glutamine (ThermoFisher Scientific).  Monocytes were seeded at 0.5 million cells/mL and differentiated into macrophages in the same medium containing recombinant human CSF-1 (100ng/mL Peprotech, Neuilly-Sur-Seine, France) during 6h to 3 days. Cells were cross-linked with addition of 1% formaldehyde directly to the culture medium for 10 min at RT with agitation. Fixation was stopped by addition of 125mM glycin during 5 min at RT with agitation and samples were then washed 2 times in ice-cold PBS before addition of SDS lysis buffer (Millipore, 10uL per 1.106 cells) supplemented with 1% protease inhibitor cocktail (Active Motif). Samples were vortexed and incubated 15 min on ice before 10 min sonication at 40W (Covaris S220, Woodingdean, UK). The chromatin immunoprecipitation was carried out using ChIP-it express kit according to manufacturer's instruction (Active Motif) with a monoclonal anti-CSF-1R antibody (sc-46662, santa cruz bioechnology). Enriched DNA from ChIP and Input DNA fragments were end-repaired, extended with an 'A' base on the 3′end, ligated with indexed paired-end adaptors (NEXTflex, Bioo Scientific, Proteigene, Saint Marcel, France) using the Bravo Platform (Agilent, Les Ulis, France), size-selected after 4 cycles of PCR with AMPure XP beads (Beckman Coulter, Villepinte, France) and amplified by PCR for 10 cycles more.
Experimental Factor: time
6
Experimental Factor: immunoprecipitate
CFMS
Experimental Factor: cell type
monocyte
Experimental Factor: stimulus
CSF-1; 100 nanogram per milliliter
Experimental Factor: disease
normal