Antibody against h3k9me2

ChIP was performed, and ChIP-enriched DNA was analysed by real-time PCR^{4 (link),9 ,19 (link)}. In brief, treated cardiomyocytes were fixed and cross-liked with 1% formaldehyde for 20 min at room temperature. After terminating cross-linking by 0.125 M glycine for 5 min, cell lysates were sonicated into chromatin fragments of approximately 400 bp in length. The samples were subjected to immunoprecipitation using 5 μg of antibody against H3K9me2 (Abcam) or nonspecific IgG control (Santa Cruz) in the presence of magnetic beads conjugated with secondary antibody. Immune complexes and input were washed and eluted with buffer. Then, ChIP-enriched DNA fragments were assayed by real-time PCR using primers close to the promoter sites on the ETS1 with GAPDH used as a control. The primer sequences were listed as follows: ETS1, (forward, 5′-AGGGTGGAGATGGGAGATGTGA-3′; reverse, 5′-AGGGTGGAGATGGGAGATGTGA-3′); GAPDH, (forward, 5′-GCCGAAGTACCCAAGGAGACC-3′; reverse, 5′-AGCAAAGGCGGAGTTACAAGG-3′).

The procedure used is similar to methods reported previously (Kyzar et al., 2017 (link); Vetreno et al., 2019 ). Briefly, basal forebrain tissue samples from vehicle- and galantamine-treated CON- and AIE-treated subjects (n = 7 subjects per group) in the galantamine restoration study were homogenized, fixed in 1.0% methanol-free formaldehyde, quenched with 1.0 M glycine, lysed with lysis buffer (1.0% [v/v] SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.0]), and chromatin sheared to fragments of < 1,000 bp on a Covaris ME220. Input DNA fractions were removed from the sheared chromatin to be processed separately and the remaining sheared chromatin was incubated overnight at 4°C with an antibody against H3K9me2 (5 μl/sample; Abcam) (see Table 1). Protein A Dynabeads (ThermoFisher Scientific, Austin, TX) were added and rotated at 4°C for 1 h followed by six washes in ChIP wash buffer. Both immunoprecipitated DNA and input DNA were eluted in 10% (w/v) Chelex by boiling at 95°C for 10 min followed by centrifugation. The resulting DNA was quantified using qPCR with SSOAdvanced Universal SYBR Green Supermix (Bio-Rad, Berkeley, CA) using primers targeted against the ChAT and TrkA promoters (see Table 2). The ΔΔCt method was used to determine fold change relative to CON and was normalized to the Input DNA fraction.