A2359

ChIP was performed using SimpleChIP Plus Enzymatic Chromatin IP Kit (#9005, Cell Signaling), following the manufacturer's instructions. Briefly, the cells were collected and cross‐linked with 1.5% formaldehyde for 20 min. After cross‐linking, DRG tissues were disaggregated into a single‐cell suspension. Cells were then lysed and the chromatin was fragmented by micrococcal nuclease to obtain chromatin fragments of 150–500 bp. Then the chromatin samples were incubated with ChIP‐grade protein G magnetic beads and antibodies against CDYL (1:100, HPA035578, Sigma), H3K27me3 (1:200, A2363, Abclonal), H3K9me2(1:200, A2359, Abclonal), H3K9me3 (1:500, A2360, Abclonal), H3K27ac (1:500, A7253, Abclonal). After reversal of protein‐DNA cross‐links, the DNA was purified using DNA purification spin columns provided in the kit. The enrichment of precipitated DNA sequences was analyzed by real‐time PCR using primers listed in Table S1, Supporting Information.

ChIP analysis was performed as described previously 20 (link). Briefly, after being crosslinked by 1% formaldehyde for 10 minutes at 37 °C, the cells were resuspended in 300 mL of lysis buffer and sonicated for 10 minutes. The supernatants were incubated with specific antibodies against UHRF1 (sc-373750, Santa Cruz biotechnology, USA), KLF6 (sc-7158, Santa Cruz biotechnology, USA), DNMT1 (sc-271729, Santa Cruz biotechnology, USA), H3K9me2 (A2359, ABclonal, USA), or immunoglobulin G control (Millipore, USA). The immunoprecipitated DNA was then purified using a DNA purification kit (QIAGEN, Germany) and subjected to PCR amplification. For re-ChIP assays, after being combined with protein A agarose beads and the indicated primary antibodies, the complexes were washed and sequentially eluted from the first ChIP by incubation with 10 mM DTT in 1× TE for 30 minutes at 37 °C. The DNA-protein-antibody complexes were then diluted 20 times with dilution buffer and subjected to a second round of immunoprecipitation with the indicated antibodies. After elution and DNA purification, extracted DNA was analyzed by PCR using primers spanning the proximal promoter regions of target genes. The PCR products were normalized to the input. The specific primers are listed in the Table S2 (Supplymentary Information).