Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) assays were conducted using a Simple-ChIP enzymatic ChIP kit (Cell Signaling Technology, Boston, MA, USA). In brief, HT22 cells in the logarithmic growth phase were incubated with 1% formaldehyde in a 10-cm cell culture dish for 10 minutes at room temperature (about 20°C), then treated with glycine for 5 minutes. Next, the cells were lysed with PMSF-containing lysis buffer, sonicated, and incubated with nuclease for 20 minutes to digest the chromosomal DNA. Appropriate DNA digestion confirmed by agarose gel electrophoresis, and a sample of the digested DNA was set aside for use as the 2% input positive control. The remaining DNA was incubated with an anti-Hoxa5 antibody (1:100, Affinity Bioscience, Beijing, China, Cat# DF4123), an anti-methylated H3K14ac antibody (5 µg/mL, Abcam, Cat# ab82501), or a negative control anti-IgG antibody overnight at 4°C on a shaker. All DNA bound to the antibodies was then purified using spin columns. The target DNA products were PCR-amplified using sequence-specific primers, and images were collected after agarose gel electrophoresis. The primers used for each PCR reaction are provided in Additional Table 2. For each PCR reaction, the corresponding input DNA was included in parallel as a positive control for PCR validation. Then, quantitative real-time PCR was performed using SYBR Premix Ex Taq II (Tli RNase H Plus) (Takara) to quantitate the DNA immunoprecipitated by the anti-H3K14ac antibody; the primers used for this reaction are provided in Additional Table 3. The results were normalized to input and are displayed as enrichment relative to the IgG control.

The primers for each polymerase chain reaction set in chromatin immunoprecipitation assay to confirm the binding sits for Hoxa5 in the promoter region of Fam3a

The primers of -1000 bp-TSS of Hoxa5 amplified with the DNA precipitated by H3K14ac to detect the percentage of relative to its corresponding input in quantitative real-time polymerase chain reaction