Chip enzymatic chromatin ip kit

A ChIP assay of liver lysates was conducted using the ChIP Enzymatic Chromatin IP Kit (#9003, Cell Signaling Technology). Liver tissues from mice fed a KD after adenovirus injection were lysed and cross-linked by incubating cells in 1% formaldehyde for 15 min at room temperature. Cross-linking was stopped by 5 min of incubation with 125 mM glycine. Chromatin was immunoprecipitated overnight at 4 °C with antibodies against NCoR1 or nonspecific IgG (Cell Signaling Technology). Data were normalized to input quantity. All primer sequences are listed in Supplementary Table 4.

Chromatin immunoprecipitation (ChIP) assay was performed using ChIP Enzymatic Chromatin IP Kit (Magnetic beads, Cell Signaling, Danvers, MA) according to the manufacturer’s instructions. Briefly, the cells were crosslinked with formaldehyde of 1% final concentration first. Then, they were washed with pre-cold PBS and collected, followed by sonication crush. The solution complexes were immunoprecipitated using the anti-PPARG2 antibody (1 : 100; catalog number sc-166731, Santa Cruz, CA) or rabbit immunoglobulin G (IgG, negative control). After that, the immunoprecipitated complexes were collected using protein G-agarose beads. The precipitates were eluted from the beads and the DNA–protein complexes were de-crosslinked at last. The DNA samples were recollected and used for PCR analysis. The PCR conditions were as follows: the holding stage keeps 95 °C for 5 min (1 cycle), the cycling stage holds 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s (35 cycles), and 72 °C for 10 min (1 cycle). ChIP primers for detailed sequences were shown in Supplementary Table S1.

Simple chromatin immunoprecipitation (ChIP) Enzymatic Chromatin IP Kit (Cell Signaling Technology, Danvers, MA, USA) was used for ChIP assays according to the manufacturer's protocol. Briefly, cells were crosslinked with 1% formaldehyde and collected in lysis buffer. Chromatin was then digested with micrococcal nuclease. Immunoprecipitation was incubated with 3 μg of anti-INSM1 antibody or normal rabbit IgG followed by immunoprecipitation with protein G agarose beads during an overnight incubation at 4°C with gentle shaking. As an input reference, 2% of the volume was removed before incubation with the antibody and stored at −20°C. The ChIP DNA was reverse crosslinked with 5 mol/L NaCl and Proteinase K and then purified. Immunoprecipitated DNA was amplified by PCR using primers, which are listed in Supplementary Table 3.

A ChIP assay of liver lysates was conducted using the ChIP Enzymatic Chromatin IP Kit (#9003, Cell Signaling Technology). Liver tissues from mice fed a KD after adenovirus injection were lysed and cross-linked by incubating cells in 1% formaldehyde for 15 min at room temperature. Cross-linking was stopped by 5 min of incubation with 125 mM glycine. Chromatin was immunoprecipitated overnight at 4 °C with antibodies against NCoR1 or nonspecific IgG (Cell Signaling Technology). Data were normalized to input quantity. All primer sequences are listed in Supplementary Table 4.

According to the manufacturer’s instructions, simple ChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, Danvers, MA, USA) was applied to ChIP assays. Briefly, cells were crosslinked with EBM-2 containing 1% formaldehyde and collected in lysis buffer. And then micrococcal nuclease was used to digest the chromatids. Immunoprecipitation was incubated with 3 mg of anti-Hoxa13 antibody (1:1000, ABcam, UK) or normal rabbit IgG followed by immunoprecipitating with Protein G Agarose Beads and stored at 4 ℃ overnight with gentle shaking. Then the DNA crosslink was reversed by 5 mol/l NaCl and Proteinase K. Finally, DNA was purified. Immunoprecipitated DNAs were amplified by PCR according to their specific primers as follows:
Control PCR1: forward 5′-TGAAAATGTGGACTAGAGCCAGA-3′;
reverse 5′-CAGACACTCCAGAACAGGGC-3′;
AGGF1PCR2: forward 5′-TCCCCGCATCCATCCTCTTA-3′;
reverse 5′-CCCATTCCCACTTCCTCCAC-3′.

The simple ChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) was used following the manufacturer’s instruction. In brief, primary antibodies (histone deacetylase 4 [HDAC4], HDAC6, or IgG antibody as a control) were coated to get immunoprecipitated DNA, which was subjected to real-time PCR (RT-PCR) using specific BDNF promoters primers (PI, PII, PIII, PIV, and PIX) (Supplementary Table S1). Then the cumulative fluorescence for each amplification was normalized to the input DNA.

A ChIP assay of liver lysates was conducted using the ChIP Enzymatic Chromatin IP Kit (#9003, Cell Signaling Technology). Liver tissues from mice fed a KD after adenovirus injection were lysed and cross-linked by incubating cells in 1% formaldehyde for 15 min at room temperature. Cross-linking was stopped by 5 min of incubation with 125 mM glycine. Chromatin was immunoprecipitated overnight at 4 °C with antibodies against NCoR1 or nonspecific IgG (Cell Signaling Technology). Data were normalized to input quantity. All primer sequences are listed in Supplementary Table 4.