Sybr green and hotstartaq dna polymerase

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.

Total RNA was isolated from the myoblasts using Total RNA kit I (Omega) following the manufacturer’s protocol. Reverse transcription to cDNA was performed using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Total RNA was quantified by Nanodrop (ND-1000), and real-time PCR was conducted using a SYBR^® Green and HotStarTaq DNA polymerase (Qiagen) on a CFX96 Touch Real-Time PCR Detection System (BioRad). Each sample was PCR amplified in triplicate. Results were analyzed by the threshold cycle (Ct) comparative method using internal controls. Myogenin primers have been previously described (AlSudais et al., 2016 (link)), while the Dag1, Rps26, and Tbp primers are designed as follows:
Dag1-F: 5′-GGT​TGG​CAT​TCC​AGA​CGG​TA; Dag1-R: 5′-CCT​GCT​GCA​GAC​ACC​TTG​AT.
Rps26-F: 5′-GCC​ATC​CAT​AGC​AAG​GTT​GT; Rps26-R: 5′GCC​TCT​TTA​CAT​GGG​CTT​TG.
Tbp-F: 5′, TCATGGA CCAGAACAACAGC; Tbp-R: 5′-GCT​GTG​GAG​TAA​GTC​CTG​TGC.