Fig. 1: Outlines of the reverse ChIP procedure. | Communications Biology

Fig. 1: Outlines of the reverse ChIP procedure.

From: Reverse Chromatin Immunoprecipitation (R-ChIP) enables investigation of the upstream regulators of plant genes

Fig. 1

The plant material was treated with formaldehyde for DNA and protein cross-linking, and nuclei were isolated using sucrose density gradient centrifugation. Then the chromatin was sonicated to shear chromatin into small fragment. A set of probes labeled with biotin was added to the sonicated chromatin, and heated at 90 °C for 2 min to denature the chromatin so that the probes can bind to the denatured DNA sequence. Hybridization was then performed to anneal probes and chromatin. After hybridization, streptavidin magnetic beads were used to affinity the probes with biotin for collecting the hybridized chromatin. After washing to remove contamination, the hybridized chromatin was eluted, and DNA-associated proteins were subjected to mass spectrographic analysis. Asterisk (*) indicates the steps that need to be studied for optimization in this study.

Back to article page