ChIP Analysis in Leaf Tissue

ChIP analysis was performed in fully expanded leaves from 4-week-old pretreated plants (50 μMAA or Mock + 0.02% tween 20 by spray). Three dat, fully expanded leaves from at least 12 different plants/treatment were included in each of the samples (following a randomized block design). Each condition was constituted by two-four different samples (two replicates were used only in BTH treatments as internal experimental control). Chromatin isolation and analysis were conducted as described in Haring et al. (2007 (link)) from 2 g of leaf tissue per sample. Chromatin immunoprecipitation was performed, using EpiQuik Chromatin Immunoprecipitation Kit (P-2002, Epigentek) with the antibody antiH3K4m3 (#07-473 Millipore). Immunoprecipitated samples were quantified by q-PCR analysis in a 7,500 real-time PCR system (Applied Biosystems), using NZYSpeedy qPCR Green Master Mix (MB22303, Nzytech) and specific primers previously reported by Jaskiewicz et al. (2011 (link)). Relative levels were calculated by the method of the reference sample, as described in Rao et al. (2013 (link)). The total amount of DNA/sample was corrected, using values of input aliquots (non-immunoprecipitated) of each sample. Finally, the values were expressed as relative rates, being “1” the average of the control, Mock. ChIP analyses were performed three times with similar results.