title: Illumina NovaSeq 6000 paired end sequencing; ChIP-Seq of H3K4me3 in primary unstimulated monocyte subsets from 2 healthy donors buffy coats
design: ChIP-Seq of H3K4me3 in primary unstimulated monocyte subsets from 2 healthy donors buffy coats
library_construction_protocol: Monocytes subsets were first enriched from mononuclear cells by negative selection using CD19-, CD56-, CD3-, CD16-microbeads and the AutoMacs system (Miltenyi Biotech), then sorted using CD45, CD14, and CD16 antibodies after exclusion of CD56+ cells and a cell sorter.  Cells were cross-linked with addition of 1% formaldehyde to the culture medium for 10 min at room temperature with agitation. Fixation was stopped by addition of 125mM glycine during 5 min at RT with agitation before washing samples twice in ice-cold PBS and adding SDS lysis buffer (Millipore, 10uL per 1.10^6 cells) supplemented with 1% protease inhibitor cocktail (Active Motif). Samples were incubated 15 min on ice before storage at -80°C. Cross-linked DNA were sonicated using 12x24 mm round bottom tube, 10 min at 40W (Covaris S220, Woodingdean, UK). 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 more cycles.
attributes.tag: Experimental Factor: cell type
attributes.value: non-classical monocyte
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: input DNA