Fig. 2

Schematic outline of the Chromatin Enrichment for Proteomics in Plants (ChEP-P) procedure. Overview of the experiment (upper panel) and key steps highlighting the changes made for plant material (lower panel). (1) Chromatin crosslinking for plant material was performed as described previously for chromatin immunoprecipitation [33]. (2) The cell lysis step was modified to suit extraction of plant proteins. (3) Chromatin enrichment was performed as described in Materials and Methods (4) SDS-PAGE gel showing the chromatin-enriched fraction during the ChEP-P procedure. (5) Samples were digested with modified trypsin and quantified. (6) For LC-MS/MS analysis, peptides were redissolved in solvent containing formic acid and acetonitrile in water. Three technical repeats were used for each of the three biological replicates . (7) Proteome Discoverer™ Software 2.2 (Thermo Fisher) with Sequest was used for the identification and label-free quantification of peptides. All peptide spectrum matches were filtered with a q-value threshold of 0.05 (5% RDR), proteins were filtered with medium confidence threshold (q-value < 0.05, 5% FDR). Adapted from Kustatscher et al. [22] with the indicated modifications