Sc 32758x

C2C12 myoblasts were crosslinked and sonicated followed by chromatin immunoprecipitation as previously described (Hamed et al., 2013 (link)). Antibodies against p300 and MyoD were purchased from Santa Cruz (sc-584x and sc-32758x). The immunoprecipitants were captured by Dynabeads protein-A, washed and eluted according to manufacturer’s protocol (Invitrogen, Waltham, MA, USA) (Hamed, Chen & Li, 2022 (link)). Chromatin DNA was reverse crosslinked at 65 °C for overnight, purified with the DNA purification kit (Qiagen, Hilden, Germany) and amplified with SYBR® Green and HotStarTaq DNA polymerase (Qiagen, Hilden, Germany) on a CFX96 or CFX384 Touch Real-Time PCR Detection System (BioRad, Hercules, CA, USA) (Hamed, Chen & Li, 2022 (link)). Each sample was amplified in triplicate PCR reactions. Purified input DNA was used to generate a standard curve for the PCR amplification of each immunoprecipitation (Hamed, Chen & Li, 2022 (link)). The abundance of immunoprecipitated DNA was quantified as the percentage relative to the input chromatin DNA (Hamed, Chen & Li, 2022 (link)). Primer pairs used for the amplification were as follows:
Hdac11: F5′-GGGTGTAGGGGGAATGGAGA; R5′-TGCCTTGAACCTGTTTCCCT. Chromosome 15 gene desert, a negative control locus: F5′-TCCTCCCCATCTGTGTCATC; R5′-GGATCCATCACCATCAATAACC.