Enrichment of H3K27ac in the promoter or exon regions of seven candidate genes was assessed with ChIP-qPCR using 0.25 ng of ChIP DNA and equivalent volume of the 10% input DNA for each reaction. ChIP-qPCR primers were designed in NCBI-Primer Blast [65 (link)], and confirmed for specificity of targeted PCR product by melt-curve analysis. The targets regions were predicted using the published tracks from H3K27ac/H3k4me2 in mouse quadricep muscle on the UCSC genome browser. Primers were:
Atf3 F1: CTAGGGCTTCAGTCTCCGGT, R1: GCGAAGACTGGAGGTGAGTT;
Bcl2l1 F: GTCTCCTTCGTCCCTTGTGG, R1: TTCCTCAGCGGATGGAAACC;
Btg2 F1: TAAAGACACCCCAGGCAAGA, R1: CTCAAGGTTTTCAGTAGGGCG;
Gemin5 F1: GGGTCTAGGCTGGGTCAGTC, R1: GGGAGTTCTGTAAGTCGCCG;
Junb F1: ATAGGGATCCGCCAGGTTGA, R1: CCGGATGTGCACGAAAATGG;
Kcnc3 F1: ACGTGCTCAACTACTACCGC, R1: CCACGTCCGTCTCGTCTATG;
Mettl11b F1: GCTCTGACTCACTTACCCAGG, R1: AGATCCCGTTGGCAGAAGAC.
Each reaction was performed in triplicate using 1X advanced low-ROX qPCR master-mix with Supergreen (Wisent), 20 µM of each primer and were run using ABI Prism 7000 Real-Time detection system (Applied Biosystems). Percent input method was used for fold change calculations.
Atf3 F1: CTAGGGCTTCAGTCTCCGGT, R1: GCGAAGACTGGAGGTGAGTT;
Bcl2l1 F: GTCTCCTTCGTCCCTTGTGG, R1: TTCCTCAGCGGATGGAAACC;
Btg2 F1: TAAAGACACCCCAGGCAAGA, R1: CTCAAGGTTTTCAGTAGGGCG;
Gemin5 F1: GGGTCTAGGCTGGGTCAGTC, R1: GGGAGTTCTGTAAGTCGCCG;
Junb F1: ATAGGGATCCGCCAGGTTGA, R1: CCGGATGTGCACGAAAATGG;
Kcnc3 F1: ACGTGCTCAACTACTACCGC, R1: CCACGTCCGTCTCGTCTATG;
Mettl11b F1: GCTCTGACTCACTTACCCAGG, R1: AGATCCCGTTGGCAGAAGAC.
Each reaction was performed in triplicate using 1X advanced low-ROX qPCR master-mix with Supergreen (Wisent), 20 µM of each primer and were run using ABI Prism 7000 Real-Time detection system (Applied Biosystems). Percent input method was used for fold change calculations.