Instrument: Illumina HiSeq 1000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: A blood sample of 30 ml was taken and peripheral blood mononuclear cells were isolated using ficoll-based density gradient centrifugation. Further isolation of CD14++ CD16- monocytes was conducted using magnetic cell sorting to deplete CD16++ monocytes and separate CD14++ monocytes. Purity of isolation was checked by flow cytometric analysis of the CD14+ cell fraction. Cells were cross-linked for 10 min using formaldehyde (final concentration 1%). Fixation was quenched by addition of glycine to a final concentration of 125 mM. After washing, cells were resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris/HCl, pH8.1, 1x concentrated protease inhibitors) and sonciated using a Branson-250 Sonicator (http.www.bransonultrasonics.com) using the microtip setting. Fragment size of the obtained chromatin was checked to be between 100 bp and 300 bp. Sheared chromatin was diluted 1/10 with dilution buffer (SDS 0.01%, Triton X-100 1%, EDTA 1.2 mM, Tris/HCl, pH 8.1 16.7 mM, NaCl 167 mM). Chromatin was precleared using a protein A/G-sepharose mixture for 1 hr at 4°C and incubated with apprpriate antibodies over night. Immune complexes were precipitated with protein A/G-sepharose and washed with low salt (SDS 0.1%, Triton X-100 1%, EDTA 2 mM, Tris/HCl, pH8.1 20 mM, NaCl 150 mM), high salt (SDS 0.1%, Triton X-100 1%, EDTA 2 mM, Tris/HCl, pH8.1 20 mM, NaCl 500 mM), LiCl buffer (LiCl 0.25 M, NP40 1%, Deoxycholat 1%, EDTA 1 mM, Tris/HCl, pH8.1 10 mM) and twice with TE. After crosslink reversal, immunprecipitated nucleic acids were purified on GFX columns (GE Healthcare). ChIP-seq library preparation using MicroPlex kit from Diagenode