GSM3005203: MNase-treated chromatin DNA_ThruPlex; Saccharomyces cerevisiae; MNase-Seq
The cultured yeast cells were collected with centrifugation at 2,000 × g for 5 min, and re-suspended in 1 ml of spheroplast buffer that contained 1 M sorbitol; 50 mM Tris-HCl, pH 7.5; and 1 mM β-mercaptoethanol. The cell suspension was supplemented with 20 µl of 10 mg/ml Zymolyase-100T (Seikagaku Kogyo, Tokyo, Japan) and incubated at 30 °C for 10 min to make spheroplasts. The spheroplasts were collected by centrifugation at 2,000 × g for 5 min and resuspended in 1 ml of MNase digestion buffer (50 mM Tris-HCl, pH 7.6; 1 mM CaCl2; 0.2% (v/v) Triton X-100) containing 1× protease inhibitor (Nacalai Tesque, Kyoto, Japan). The lysate was digested with 50 units of micrococcal nuclease (Takara Bio Inc.). After incubation at 37 °C for 10 min, the digestion was stopped by adding 100 µl of 500 mM EDTA. The reaction mixture was then supplemented with 100 µl of 10% (w/v) SDS and 25 µl of 20 mg/ml Protease K (Qiagen), and incubated at 50 °C for 1 hr. Following phenol and phenol/chloroform extraction, nucleic acids were collected by isopropanol precipitation. Two library construction methods were used. The one used ThruPlex kit according to manufacturees instruction, the other is based on TCS ligation.
GEO
GSM3005203
GEO Accession
GSM3005203