application example

Chromatin immunoprecipitation (ChIP). Chromatin immunoprecipitation was performed as described (Bowler et al. (2004), Plant Journal) with modifications. Sonic and IP buffer were prepared as described (Kaufmann et al. (2010)), Nature Protocols). Chromatin pellet was sonicated at 4 °C with a Diagenode Bioruptor set at high intensity for 10 min (30 sec on, 30 sec off intervals) to obtain 250-500 bp DNA fragments. Chromatin (20 µg) was immunoprecipitated with a RNA Pol II antibody (2µg/IP)and Dynabeads®Protein G (Life Technologies). Reverse corsslinking and elution of DNA fragments were performed using Chelex (Biorad) at 95°C, 10 min as described (Rowley et al. (2013), Methods). Sample without antibody was used to determine the nonspecific background pulled-down directly by the beads. Relative abundance of regions of interest in immunoprecipitated DNA was measured by real-time PCR. Red lines indicates amplified region. Values on the charts are shown as the mean ± SD level (% of input) of RNA Pol II on GAPDH gene (A) and MIR168a (B) gene. Data comes from three independent experiments.

Courtesy of MSc Jakub Dolata, Adam Mickiewicz University, Poland