Simplechip plus enzymatic chromatin ip kit

Chromatin immunoprecipitation (ChIP) were performed using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology, 9005) according to enclosed chromatin immunoprecipitation protocol. Briefly, cells were incubated with 1% formaldehyde for 10 min at room temperature to cross-link proteins to DNA, nuclei were digested with micrococcal nuclease and sonication. Cross-linked and digested chromatin was immunoprecipitated using indicated antibodies. Immunoprecipitated chromatin was incubated with 5 M NaCl and Proteinase K at 65 °C to reverse cross-links and followed by DNA purification. DNA was quantified by real-time PCR using respective primers. Primer sequences were attached in Table S1.

Mice treated with either VPA or saline for 7 days were anesthetized deeply with isoflurane and the forebrain was rapidly dissected and stored at –80 °C on PND15. Approximately 25 mg of the forebrain tissue was minced on ice using a scalpel and incubated in 1.5% formaldehyde solution for 20 min at room temperature to cross-link the proteins to the DNA. The SimpleChIP Plus Enzymatic Chromatin IP Kit (#9005, Cell Signaling Technology, Tokyo, Japan) was used in the subsequent assay procedure. The cross-linked tissue was sonicated with a Bioruptor (UCD-250, Cosmo Bio, Tokyo, Japan) to reduce the size of DNA fragments to 150 to 900 bp and chromatin was immunoprecipitated using rabbit polyclonal antibody to MBD1 (1:50; sc-25261, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal antibody to MBD2 (1:50; ab38646, Abcam), or rabbit polyclonal antibody to MeCP2 (1:500; #07-013, Millipore). ChIP-Grade Protein G magnetic beads were used to enable the rapid isolation of protein/DNA complexes from the crude chromatin mixture. After reversal of cross-links with 5 M NaCl and proteinase K, 1/25 of DNA purified from ChIP was subjected to quantitative PCR (qPCR) using a primer set designed to target the mouse Gad1 promoter region (forward: 5′-CACACACCCTCCTTTCTGGT-3′; reverse: 5′-GAAGGGAGAGATCCGGAGAG-3′).

The chromatin immunoprecipitation (ChIP) assay was conducted by using SimpleChIP^® Plus Enzymatic Chromatin IP Kit with agarose beads (Cell Signaling Technology, MA, United States) following the supplier’s protocol with minor modification. In brief, LUAD cell lysates were treated with 1% formaldehyde and glycine, followed by chromatin digestion using micrococcal nuclease. Afterward, 5 μg p53 antibody (sc-126, TX, United States) was added into cell lysates and incubated overnight. After washing, the DNA fragment pulled down by p53 antibody was eluted for PCR analysis.

The STAT5b binding motifs in the 4 kb nucleotide sequence upstream of the eGFP translation start site in the pCAG-eGFP Tg mouse line were analyzed by the JASPAR database. Two predicted STAT5b binding motifs, one that exhibited sexually dimorphic CpG methylation in the R4 region and another that had the highest prediction score in the R5 region, were further analyzed by a SimpleChIP^® Plus Enzymatic Chromatin IP kit (Cell Signaling Technology, Danvers, MA, USA), according to the manufacturer’s instructions. The STAT5 antibody (Cell Signaling Technology) was used for the immunoprecipitation, and normal rabbit IgG was used as a negative control. The DNA fragments from the ChIP were analyzed by Q-PCR. The signal of the DNA fragments from the 2% input chromatin was used as a loading control, and the values were calibrated relative to the IgG control. The Q-PCR primers are shown in Supplementary Table S1.

ChIP assays for HEC251 cells were performed with SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling) according to the manufacturer’s protocol. Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature, then treated with micrococcal nuclease to obtain fragments with a length of approximately 150–900 bp. The fragmented chromatin was subjected to immunoprecipitation with antibodies against TCF7L2, EP300 RNA polymerase II, H3K27ac, and SOX4. Normal rabbit IgG was used as negative control. The purified immunoprecipitated chromatin, input chromatin, and mock-IP (IgG) was subjected to PCR amplification of the candidate SNP site by using oligonucleotides primers (S3 Table). The binding affinities were measured by real-time quantitative PCR with the KAPA SYBR FAST qPCR kit (KAPA Biosystems) on the 7900HT sequence detection system (Applied Biosystems). The binding affinity in the immunoprecipitated chromatin was normalized to that in the corresponding input chromatin as follows: $2^{-\left(C{t}_{IP}-C{t}_{Input}\right)}$ . The enrichment of the normalized binding affinity for the candidate SNP site or positive control region was normalized by dividing by that for negative control region. We used MYC promoter and α satellite repeat element as positive and negative control regions, respectively (S3 Table). Details on the antibodies are shown in S4 Table.

ChIP assays of cultured MCF-7 were performed using the SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology, Davers, MA). Anti-REST ChIPAb + antibody (Millipore, #17–641), Anti-Histone H3 (Cell Signaling Technology, #4620), RNA Polymerase II (Millipore, #05–623), H3K27Me3 (Millipore, #07–449), and Rabbit IgG (Cell Signaling Technology, negative control) were used according to manufacturer’s protocol. PCR primers spanning the conserved RE1 elements in MMP24 and CEMIP were designed based on the genomic sequence. The primer sequences are found in Additional file 3. For quantification, ImageJ software was used to measure band intensities and the samples were normalized to their respective inputs. Original gel images are included in Additional file 11.