Fig. 2

Immunoselection compatibility of chromatin isolated using different extraction strategies. Chromatin from normal colon FFPE samples at different times of fixation, extracted following the different strategies described above, was immunoprecipitated with an anti-H3K4me3 antibody. After immunoselection, chromatin was de-crosslinked and the DNA purified and fluorimetrically quantified (a). Input fractions were also purified and the percentage of enrichment by the antibody compared to the input was calculated (b). Transcriptionally active (VCL and GAPDH) and inactive (HAPLN1 and COL2A1) promoter regions were amplified by real-time qPCR to evaluate the specificity of the immunoselection. H3K4me3 enrichments are expressed as percentage of bound compared to the input (c). Mock (no antibody) control did not produce amplification. Std: standard PAT-ChIP, 18 pulses of sonication of 5 s at 85% of amplitude; A: 54 pulses of sonication of 5 s at 75% of amplitude; B: 54 pulses of sonication of 5 s at 65% of amplitude; LRC: condition in which the sample was subjected to limited reversal of crosslinking, 3 pulses of sonication of 30 s at 40% of amplitude. **P < 0.01 with respect to standard condition for each time of fixation by one-way ANOVA with Tukey’s HSD. All the experiments were conducted in triplicates