ChIP Assay for Hes5, SIRT1, and Histone Modifications

Chromatin immunoprecipitation (ChIP) assays were performed essentially as described before [57 (link), 73 (link)–90 ]. In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ~200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-Hes5 (Abcam, ab194111), anti-SIRT1 (Santa Cruz, sc-74504), anti-acetyl H3 (Millipore, 06-599), anti-acetyl H4 (Millipore, 06-866), or pre-immune IgG. Precipitated DNA was amplified with the following primers: #1, 5’-AGAGTGAGACAGGGCCAAGAC-3’ and 5’-AAACCGAAATTGCTCAACACAC-3’; #2, 5’-AAACCCACAACGTATTA-3’ and 5’-ATGCAGCAATGAACAAC-3’.