Illumina HiSeq 2000 sequencing; ChiP-seq of H3K4me3 in primary unstimulated monocytes from 3 healthy donors buffy coats and from 4 CMML patients
ChiP-seq of H3K4me3 in primary unstimulated monocytes from 3 healthy donors buffy coats and from 4 CMML patients
Peripheral blood mononucleated cells (PBMC) were obtained from healthy donor buffy coats (Etablissement Français du sang, Rungis, France) and from CMML patients, separated on Pancoll (Pan-Biotech, Dutscher, Brumath, France). Peripheral blood classical monocytes were sorted with magnetic beads and the AutoMacs system (Miltenyi Biotech, Paris), using the monocyte isolation kit II (Miltenyi Biotech, Paris, France). 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 polyclonal anti-H3K4me3 antibody (39159, Active Motif). 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: disease
normal
Experimental Factor: immunoprecipitate
H3K4Me3