Magnify chip

ChIP experiments on MuSC-derived myoblasts were performed on chromatin extracts according to the manufacturer's protocol (MAGnify ChIP; Life Technologies) by O.N. incubation with 3 μg of immobilized anti-H3K9me2 (Abcam ab1220; RRID:AB_449854) or rabbit IgG (14-4616-82; RRID:AB_2865072) antibodies. A standard curve was generated for each primer pair testing 5-point dilutions of input sample. Fold enrichment was quantified using qRT-PCR (SYBR Green; Qiagen) and calculated as a percentage of input chromatin (% Inp). Data from GAP-SCR vs GAP‐REW conditions represent the mean of six independent experiments ± SEM. For G9a ChIP experiments on proliferating C₂C₁₂, crosslinking was performed by adding DSG (di-succinimidyl glutarate; Santa Cruz) at a final concentration of 2 mM for 45 min at RT. Then, formaldehyde (Sigma) was added to culture medium to a final concentration of 1% for 10 min at RT and stopped by glycine to a final concentration of 0.125 M. Chromatin was extracted as described in Mozzetta et al., 2014 (link) and immunoprecipitated with 5 μg of G9a (Abcam, ab185050; RRID:AB_2792982) or rabbit IgG (14-4616-82; RRID:AB_2865072) antibodies carried out overnight at 4°C. Sequences of the oligonucleotides used for ChIP analyses are reported in Supplementary file 1.

ChIP assays were performed using MAGnify™ ChIP according to the manufacturer’s instructions (Thermo Fisher Scientific). In brief, C2C12 cells were incubated with DMP-PYT for 24 h and fixed in formaldehyde for 10 min. Cell suspensions were sonicated using High power sonication under the optimized condition (20 cycle; 30 pulses of 30 s each with a 30-s rest on ice between pulses). Cell lysates were incubated with anti-TCF4 antibody and chromatin-immune complexes were captured to magnetic beads and eluted in PBS after washing. Quantitative real-time PCR was performed using primer sets detecting TCF4 binding elements in distal promoter of BMP2 gene. The following primers were used for ChIP-PCR: 5′-gacctctacagctctagaaacag-3′ and 5′-cattcaggagccttcatagtacc-3′.