Magna chip protein g magnetic beads

Chromatin immunoprecipitation (ChIP) was performed using a Magna ChIP™ Protein G Magnetic Beads (Millipore, MA, USA) (16-662) according to the manufacturer’s instructions. EC109 cells were cultured in 100-mm dishes and treated with DMSO, SFE (20 μΜ), or with a combination of SFE and LPS (0.3 mg/L) for 48 h. After fixed with 1% formaldehyde for 10 min and washed with cold phosphate buffer saline (PBS), cells were lysed using cell and nuclear lysis buffer and sonicated on ice using a Sonics Vibra-Cell processor (Sonics & Materials Inc., Newtown, CT, United Kingdom) to generate DNA fragments. Approximately 2% of the suspension was removed to determine the input quantity of DNA. Then chromatin was immunoprecipitated by incubating overnight at 4 °C with protein G magnetic beads and the following antibodies: 3 μg rabbit anti-NF-κB p65 (8242, Cell Signaling Technology) and normal rabbit IgG (2729, Cell Signaling Technology). The precipitated DNA-protein complexes were washed with wash buffer and eluted in elution buffer. RNA was digested with RNase A for 30 min at 37 °C, and proteins were digested with Proteinase K for 2 h at 45 °C. After DNA purification, the resulting DNA was analyzed by qPCR and normalized by total chromatin (input). Primers specific to the predicted binding sites of p65 in TNFAIP3 and PLAU are described in Supplementary Table S1.

100 pmole native mononucleosome extract was mixed with 2 μg antibody (H3K4me3 - Ab8580, lot# GR56122-1; H3K9me3 - Ab8898, lot# GR102573-1; normal rabbit IgG - sc2027) and incubated with 20 μL Magna ChIP Protein G magnetic beads (Millipore) in buffer N (30 mM HEPES, 150 mM NaCl, pH 7.4, 0.01% NP-40, 5 mM EDTA) under constant rotation at 4°C overnight. Bound chromatin was further washed with buffer N three times and eluted twice with 150 μl elution buffer containing 1% SDS.

AGS cells were infected with H. pylori at an moi of 50 for 3 h, and were crosslinked with 1% formaldehyde for 10 min and then quenched in 0.125 M glycine. The crosslinked chromatin was fragmented by sonication. The lysate were subjected to IP using corresponding antibodies as indicated (anti-KDM4B, anti-c-Jun, anti-H3K9me3, and rabbit IgG) and Magna ChIP Protein G Magnetic beads (Millipore) at 4 °C overnight. The ChIP complexes were eluted by elution buffer [50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% SDS] and quantified by qRT-PCR (primer sequences listed in Table S3). Fold enrichment was calculated by the ∆∆Ct method.

ChIP experiments were performed as previously described [22 (link)]. Briefly, after cross-linking with 1% formaldehyde, cells were lysed and chromatin sonicated with Bioruptor® Pico sonicator (Diagenode SA, Ougrée, Belgium) then precipitated with Magna ChIP™ Protein G Magnetic Beads (16–662, Millipore, Burlington, Massachusetts, USA) and the appropriate antibody (Additional file 2: Table S3). The immunoprecipitated DNA fragments were analyzed by qPCR, see Additional file 2: Table S1 for primers sequences. For each experiment, a chromatin amount corresponding to 1% of chromatin used for immunoprecipitation was kept as input control. Each qPCR value was normalized over the appropriate input control and reported in graphs as input %. (qPCR value/input value × 100).