(A) Chromatin was prepared from embryonic day 11.5 (E11.5) dissected limb buds (distal, proximal, anterior, and posterior) and from 14Fp cells 18, 24, and 30 hr after trichostatin A (TSA) treatment, 24 hr treatment with DMSO was used as control (ctr). Shown are results from chromatin immunoprecipitation (ChIP) analysis using anti-H3K27ac antibody. Enrichment of H3K27ac at the 5’ spatiotemporal (5’ST) region was detected by quantitative PCR (qPCR) and represented as mean of fold enrichment/background (IgG) ± SEM over three biological replicates. (B) Quantitative reverse transcriptase PCR to detect the expression of Shh in embryonic cell lines at E11.5 from the limb (14Fp) and the mandible (MD) after TSA treatment. MDs do not express Shh in response to the treatment. The Shh levels were evaluated relative to control and normalized to Gapdh levels. (C) ChIP of 14Fp and MD cell line using antibodies to two different histone modifications (H3K4me1 and H3K27ac) analysed by qPCR. Low enrichment of H3K4me1 with the 3’ long-range (3’LR) oligos set was observed in MD in comparison to 14Fp and no enrichment of H3K27ac with the oligos set 5’ST was observed after TSA treatment. (D) Gel with three 3C samples for the 4C experiment (ctr, 18 hr TSA, and 24 hr TSA). Undigested and HindIII digested DNA after crosslinking was run on a 0.6% agarose gel and appears as a high molecular weight smear running from roughly 12 to 4 kb showing that they were all efficiently digested. (E) Final 3C templates run as one tight band above 10 kb in size on 0.6% agarose gel for the ctr, 18 hr TSA, and 24 hr TSA ligated samples. On the first lane as control a digested template is loaded. (F) Final 3C libraries are redigested with MluCI and the products were run on a 1.2% agarose gel appearing as smear between roughly 0.3 and 1 kb. As control the ligated products are run together with the samples.