GSM1692782: HB_DHS; Mus musculus; DNase-Hypersensitivity
At least 1x 10^6 up tp 3x 10^6 freshly sorted and thoroughly counted cells were resuspended at 3x 10^6 cells per 100 µl DNase I buffer (60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 10 mM Tris pH 7.4, 0.3 M sucrose), mixed with an equal volume of DNase I NP-40 buffer (60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 10 mM Tris pH 7.4, 0.3 M sucrose, 0.4% NP-40, 2 mM CaCl2) with suitable 2-fold concentration of DNase I enzyme (final concentration ranging between 15 U and 260 U) and incubated at 22°C for 3 min. The reaction was stopped by addition of 2x volume of lysis buffer (300 mM NaAcetate, 10 mM EDTA pH 7.4, 1% SDS, 1 mg/ml proteinase K) and samples were incubated over night at 45°C. After phenol-phenol/chloroform-chloroform extraction DNA was precipitated with 2x volume of 100% ethanol, 10 µg of digested DNA were run on a 1.2% agarose gel in TAE buffer, 50 - 300 bp fragments were size-selected and purified using Qiagen MinElute gel extraction kit. Before library preparation the DNase I digested and size-selected material was validated by realtime PCR analysis. For library preparation ChIP material was end-repaired, respective adaptors were ligated and fragments were PCR-amplified with 9 to 18 cycles. Size-selection and adaptor-dimer removal was done via gel purification or Ampure bead purification (for SOLiD libraries only). Libraries were validated using realtime PCR for known target/accessible sites, quality was assessed by Agilent Technologies 2100 Bioanalyser and in case of multiplexing the quantity of libraries was measured by the Kapa Library Quantification Kit.
GEO Accession
GSM1692782