Chromatin immunoprecipitation (ChIP) assay

The ChIP assays were conducted using the EZ ChIP Kit (Upstate/EMD Millipore, Darmstadt, Germany) as described.¹³ The homogenization solution from DRG was crosslinked with 1% formaldehyde for 10 min at room temperature. The reaction was terminated by the addition of 0.25 M glycine. After centrifugation, the collected pellet was lysed by SDS lysis buffer with protease inhibitor cocktail and sonicated until the DNA was broken into fragments with a mean length of 200 to 1000 bp. The fragment sizes were verified by gel electrophoresis (data not shown). After the samples were precleaned with protein G agarose, they were subjected to immunoprecipitation overnight with 2 µg of rabbit antibodies against G9a (Abcam, Cambridge, MA), CREB (Abcam), and H3K9me2 (Abcam), or with 2 µg of normal rabbit serum overnight at 4℃. Input (10%–20% of the sample for immunoprecipitation) was used as a positive control. The DNA fragments were purified and identified using PCR/Real-time PCR with the primers listed in Table 1.