title: GSM1479704: DNase ZT 14 WT; Mus musculus; DNase-Hypersensitivity
library_construction_protocol: DNase1-seq protocol: Mouse liver nuclei were prepared as described in (Tian and Schibler 1991). Freshly prepared nuclei were suspended in ice-cold Ψ-buffer (11 mM KPO4 pH 7.4, 108 mM KCl, 22 mM NaCl, 5mM MgCl, 1 mM CaCl2, 1 mM DTT) and pelleted. 5x10^6 nuclei were suspended in 200 μl of Ψ-buffer supplemented with 0.2% of NP40 and 1 u/ml of DNaseI (DPFF Worthington Biochemical Corporation). DNase1 digestion was performed for 6 minutes in room temperature and the reaction was stopped by adding 200 μl of lysis buffer (50mM Tris-HCl pH 8, 20 mM EDTA, 1% SDS, 200 μg/ml proteinaseK). Protease digestion was performed overnight at 55 oC. RNaseA (100 μg/ml) was then added and samples were incubated at 37 oC for an hour. DNA was then extracted twice with phenol-chloroform and precipitated with isopropanol in the presence of 0.5 M NaCl. DNAs were dissolved in 5 mM Tris-HCl pH 8.DNAs from 4 animals were pooled and 75 μg of DNA was loaded on 11 ml 10%-50% sucrose gradient in STE buffer (1M NaCl, 20 mM Tris-HCl pH 8, 5 mM EDTA) and centrifuged at 30000 rpm for 16 hours at 20 oC (SW 40 Ti rotor, Beckman Coulter Inc). The sucrose gradients were then fractionated and DNA was precipitated by two volumes of ethanol in the presence of 5 μg of glycogen. Fractions containing DNA sized around 300bp were pooled and used for Illumina library preparation. Libraries for ultra-high-throughput sequencing were prepared with the ChIP-Seq DNA sample kit (Illumina) as recommended by the manufacturer.
attributes.tag: GEO Accession
attributes.value: GSM1479704