Ez magna chip assay kit

Chromatin immunoprecipitation (ChIP) assays were performed using the EZ-Magna ChIP Assay Kit (Merck Millipore, Darmstadt, Germany) as previously described [20 (link)]. Briefly, hBMSCs were first crosslinked with 1% formaldehyde, collected, lysed, and sonicated to shear DNA. Then the DNA–protein complexes were isolated with antibodies against YY1 (Cell Signaling Technology) and normal rabbit IgG (Merck Millipore) as a negative control at 4 °C overnight. The next day, the protein-DNA complexes were eluted and de-crosslinked. The purified DNA was quantified by qRT-PCR and relative enrichment was calculated as the amount of amplified DNA relative to values obtained from immunoprecipitation using normal IgG. Primers are listed in Additional file 1: Table S3.

Chromatin immunoprecipitation (ChIP) assays were performed using an EZ-Magna ChIP assay kit (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s instructions. hBMSCs were seeded in 10-cm dishes, and cells were cross-linked with 1% formaldehyde after they reached 100% confluency. The cell lysate was then sonicated into DNA fragments, and the DNA–protein complexes were isolated using antibodies against DEPTOR (Merck Millipore) and isotype IgG (Cell Signaling Technology) at 4 °C overnight with constant rotation. Unbound substances were removed using elution buffer. After reversing the cross-link, the DNA was purified using spin columns, and used for quantitative ChIP-qPCR with a 7500 Real-Time PCR Detection System (Applied Biosystems). The primers specific for the MEG3 promoter are presented in Additional file 1: Table S1. The data were calculated using the 2^–ΔΔCt relative expression method. Relative enrichment was then analyzed as the amount of amplified DNA normalized to the input and relative to values obtained from immunoprecipitation with isotype IgG.

ChIP assays were performed using the EZ-Magna ChIP assay kit (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s instructions. Briefly, cells were washed with PBS and cross-linked with 1% formaldehyde for 10 min. Chromatin was sonicated on ice to generate chromatin fragments of 200–1000 bp, and then immunoprecipitated with each antibody. Antibodies for acetylated histone H3 (AcH3, Merck Millipore), tri-methylated histone H3K4 (H3K4me3, Merck Millipore), and isotype IgG (negative control, Cell Signaling Technology) were used in the immunoprecipitations. The immunoprecipitated materials were then washed extensively, and cross-linking was reversed. DNA from the eluted chromatin was purified by phenol extraction and ethanol precipitation. Input control DNA or immunoprecipitated DNA was determined by quantitative real-time PCR. Standard curves were constructed using a pool of input samples, and each ChIP sample was normalized to its respective input. We designed primers to separately amplify five regions in the FABP4 promoter region. The primer pairs used for ChIP assays are shown in Supplementary Table S1.

ChIP assay for PPs was performed with an EZ‐Magna CHIP assay kit (17‐10086, Merck) according to the manufacturer's instructions. Briefly, PPs were harvested, cross‐linked with 1% formaldehyde, lysed, and then sonicated. The DNA‐protein complexes were then isolated with appropriate antibodies which were listed in Table S2 (Supporting Information). The primers were designed according to the promoter sequences of CDH1 or HES1. The sequences of primers are shown in Table S3 (Supporting Information).