HiChIP for human Monocyte, classical - 1_S1_T2_HICHIP_H3K27AC_CLASSICAL_MONOCYTES
HiChIP was performed as described previously with some modifications (Mumbach et al., 2017). Briefly, cells were crosslinked with 1% formaldehyde and flash frozen in liquid nitrogen. Fixed cells were lysed to obtain nuclear fraction, and then chromatin was digested in intact nuclei using 200 U of the 4-base cutter MboI (New England Biolabs), and restricted ends re-ligated as described (Mumbach et al., 2017). Pelleted nuclei was dissolved in 130 µl nuclear lysis buffer (50 mM Tris-HCl, pH 7.5, 10 mM EDTA and 1% SDS) and were sonicated using a Covaris S220 for 4 minutes with the following settings: fill level 10, duty cycle 5, peak incidence power 105, cycles per burst 200. Sonicated chromatin was diluted 10 times in ChIP Dilution Buffer (50 mM Tris-HCl, pH 8, 167 mM NaCl, 1.1mM EDTA, 0.55 mM EGTA, 0.11% Na-deoxycholate, 1.1% Triton X-100  and 1X protease inhibitors) and immunoprecipitation was done overnight at 4°C by incubating 2.5 ml of H3K27ac antibody (C15410196; Diagenode) precoated on 25 ml protein A coated magnetic beads (Thermo Fisher Scientific). Immunocomplexes were captured and washed three times for 5 min each with RIPA buffer (10 mM Tris-HCl, pH 8, 140 mM NaCl, 0.1% SDS, 0.1% Na-deoxycholate, 1% Triton X-100, 0.5 mM EGTA and 1 mM EDTA), high-salt buffer (50 mM Tris-HCl, pH 8, 500 mM NaCl, 0.1% SDS, 0.5% Na-deoxycholate, 1% Nonidet-P40 and 1 mM EDTA), LiCl buffer (50 mM Tris-HCl, pH 8, 250 mM LiCl, 1 mM EDTA, 1% Nonidet-P40 and 0.5% Na-deoxycholate), and low-salt buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA, 50 mM NaCl). Beads were resuspended in TE (10 mM Tris-HCl, pH 8, 1 mM EDTA), transferred to fresh tubes, captured and then resuspended in 200 µl of elution buffer (50 mM NaHCO3 and 1% SDS). Samples were treated with 10 µg of RNase A (Thermo Fisher Scientific) for 30 min at 37°C and then with 2 µL of 20 mg/ml proteinase K (Thermo Fisher Scientific) for 1 h at 55°C, and incubated overnight at 65°C. DNA was purified using affinity columns (Zymo Research) and eluted in 20 µl of DNA elution buffer (10 mM Tris-HCl). Adapters were ligated to DNA using NEBNext® Ultra™ DNA Library Prep Kit (New England Biolabs) according to manufacturer's protocol. Streptavidin C-1 beads were used to capture biotinylated DNA according to manufacturer's protocol  and resuspended in 20 µl of DNA elution buffer (10 mM Tris-HCl). To generate the sequencing library, PCR amplification of the DNA were performed while the DNA was still bound to the beads. Purified HiChIP libraries were size selected to 300-800 bp using AMPure XP beads (Beckman Coulter Life Sciences) according to manufacturer's protocol and subjected to 2 x 50 bp paired-end sequencing on Illumina HiSeq 2500 or NovaSeq6000 (Illumina).