Fig. 3

Application and validation of EPAT-ChIP. Chromatin was extracted from an archival invasive breast carcinoma sample by both the standard PAT-ChIP procedure (Std) and the new LRC-based procedure (LRC). The amount of extracted chromatin was estimated by fluorimetric quantitation of purified DNA after complete de-crosslinking (a) and chromatin fragmentation was evaluated by electrophoretic separation on 1.3% AGE followed by SYBR Gold staining of purified input DNA (b). Chromatin was then subjected to immunoselection with an anti-H3K4me3 antibody, de-crosslinked, and the DNA purified and quantified (c). Input fractions were also purified and the percentage of enrichment with respect to the input was calculated (d). Transcriptionally active (VCL and GAPDH) and inactive (HAPLN1 and COL2A1) promoter regions were amplified by real-time qPCR (each sample amplified in triplicate) to evaluate the specificity of the immunoselection. H3K4me3 enrichments are expressed as percentage of bound respect to the input (e). Mock (no antibody) control did not produce amplification. **P < 0.01 with respect to standard condition by Student’s t test