GSM8265648: Monocytes, control plasmid (pcDNA), S100A9, ChIP, rep2; Homo sapiens; ChIP-Seq
Monocytes were pelleted at 400 g at 4°C and washed with ice-cold PBS. Protein-protein interactions were cross-linked using 1.5 mM disuccinimidyl suberate (DSS; ThermoFisher, #21655), 1.5 mM ethylene glycol bis (EGS; ThermoFisher, #21565) and 1.5 mM disuccinimidyl glutarate (DSG; ThermoFisher, #20593) for 45 min in the dark with agitation at room temperature. ChIP was performed according to manufacturer's instructions (Cell Signaling, #9005) with volumes adjusted for correct concentration during protein-DNA cross-linking. 1 µg of S100A9 antibody (Cell Signaling, #72590) or control IgG (Nordic-Mubio, #RIgG) were used per IP. Immunoprecipitation was validated by immunoblotting for S100A9 using a sample taken prior to chromatin elution from antibody/protein G magnetic beads. After DNA purification (New England Biolabs, #T3010L) DNA libraries (Cell Signaling, #56795 & #47538) were prepped using equal volumes of eluted DNA. Library samples were quantified using Qubit 4.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and library profiles were checked with DNA Kit on Agilent 5600 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA). The sequencing libraries were multiplexed and loaded onto the Illumina NovaSeq 6000 instrument according to manufacturer's instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration. Image analysis and base calling were conducted by NovaSeq Control Software v1.6 on the NovaSeq instrument.