*4.8. Relative Accessibility of Chromatin and DNA*

The chromatin was isolated from 1 g of freeze-ground green tissue by ChromaFlash Plant Chromatin Extraction Kit (EpiGentek, Farmingdale, NY, USA) according to a protocol provided by the manufacturer. The sonication stage was set for the QSonica 700 (QSonica, Newtown, CT, USA) instrument and performed with the following parameters: Amplitude–25%, Process time–3 min 20 s, Pulse ON–20 s, Pulse OFF–30 s. Isolated chromatin was divided into portions (approximately 300–400 ng per aliquot), snap-frozen in liquid nitrogen, and stored at −80 ◦C.

Digestion of chromatin and DNA was performed. First, the chromatin was digested with the following FastDigest enzymes: AatII and PvuI (Thermo Scientific, Waltham, MA, USA). The restriction enzymes were defined by in silico analysis. Sites of digestion for AatII and PvuI are located in the nucleosomes, where DNA wraps around nucleosomes. After digestion with restriction enzymes (15 min, 37 ◦C) and enzymes' inactivation (AatII, PvuI –80 ◦C, 5 min), the samples were incubated with Proteinase K (Qiagen, Hilden, Germany) and RNase A (Qiagen, Hilden, Germany) at 37 ◦C for 1 h. Simultaneously, chromatin samples were incubated first with Proteinase K and RNase A, in order to release the DNA from the nucleosome. The DNA was digested with restriction enzymes in an analogous manner as chromatin was cut. Undigested control samples were also prepared. Total DNA was purified from each sample with phenol/chloroform/isoamyl alcohol (PCI-25:24:1, *v*/*v*), mixed vigorously and centrifuged (10,000× *g*, for 5 min, at room temperature (RT)). The upper phase was transferred into a new tube and DNA was precipitated by adding 0.5 mL of cold isopropanol. In order to facilitate precipitation, 2 µL (20 µg) of linear polyacrylamide was added (5 mg of acrylamide was dissolved in 200 µL of water, 1 µL of 10% APS and 1 µL of TEMED were added, left to polymerase overnight at RT, 2.5 volumes of ethanol were added, centrifuged at 12,000× *g*, 5 min, dried and re-suspended in 500 µL of sterile water). After 10 min of incubation on ice, the samples were centrifuged 12,000× *g*, for 15 min, at 4◦C. The DNA pellet was washed with 0.5 mL of cold 70% (*v*/*v*) ethanol and centrifuged at 12,000× *g* for 10 min at 4 ◦C. The dry pellet was re-suspended in 40 µL of sterile water.

The relative accessibility of chromatin/DNA was assessed by the real-time PCR method. During the reaction, the region surrounding motif CCGG located at +1273 was amplified. The selection of this region was preferred due to the methylation status of the particular motif and the location of the sites of cutting by AatII and PvuI restriction enzymes. The real-time PCR reaction was performed similarly to the gene expression analysis. The quantity of measured DNA was calculated in order to estimate the relative accessibility of chromatin/DNA to the restriction cut. The actin gene was used as the reference. The results were presented as the relative chromatin/DNA accessibility in comparison to the undigested control, set as 1.
