title: GSM8190411: ZNF1_Mutant_MNase; Saccharomyces cerevisiae; MNase-Seq
library_construction_protocol: Cultures were cross-linked with 1% formaldehyde at room temperature (RT) for 30 min. and quenched with 0.125 M glycine at RT for 5 min. to neutralize the formaldehyde. Cells were centrifuged, washed withwater, and treated with Buffer Z (0.56M sorbitol, 50mM Tris) with 0.5 mL of 10mg/ml zymolyase at 25°C for 30 min. Extracts were centrifuged at 1500 rpm for 6 min. at 4°C and resuspended in NP buffer (1M sorbitol, 50mM NaCl, 10mM Tris pH 7.4, 5mM Mgcl2, 1mM CaCl2) containing 250mM spermidine, 0.007% b-ME, and 0.075% NP-40. In separate tubes, MNase was aliquoted as follows: 4 µL, 2 µL, 1 µL, 2 µL of 1:4 in water, and 1 µL in 1:4 water. 400 µL of extract were added to each MNase aliquot and incubated for 20 min. at RT. After digestion, 100 µL stop buffer (5mM SDS, 1mM EDTA) and 10 µL of 10mg/ml Proteinase K were added. Samples incubated O/N at 65°C. To recover DNA, samples were phenol:chloroform extracted and precipitated with 400 µL of isopropanol. DNA was treated with 0.1mg/ml RNaseA to remove RNA. Illumina sequencing libraries of MNase-treated DNA were prepared using 500 ng of DNA as previously described (Henikoff et al 2011).