FastDigest PvuI

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	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.

Protocol tips

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.

Publication protocol

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.

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Manufacturer protocol

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