GSM1199553: DHS_Ter119_Positive; Mus musculus; DNase-Hypersensitivity
DNase I assays were performed using ,56107 cells. Nuclei from mainly terminally differentiated erythroblast (,60–80% cells) were digested with eight different concentrations of DNase I (0ice,037,0.5,1,2,4,8,16,32,64mlof10U/mlDNaseIin1ml reaction volume) at 37uC (except 0ice) until the sample containing 16 ml reached water-like viscosity. The digestions were stopped instantly by transferring the tubes to an ice box followed by transferring the reactions into lysis buffer. DNA was extracted using a phenol/chloroform mix and ethanol precipitated. Since DHS represent a spectrum of signal intensities, and the optimal sample should include material from different DNase I concen- tration digestions, the mid phase of digestions were selected for library preparation by qPCR (see below). Based on the principle that DNase I may cut more than once across DHS, generating small DNA fragments that can be amplified. Samples were quantified using the High Sensitivity Qubit system (Invitrogen) and distribution of fragments was determined using Agilent High Sensitivity DNA Kit and a 2100 Bioanalyzer Instrument system (Agilent). DNA (2–4 mg) was blunt- ended with T4 DNA Polymerase (NEB), adenine overhangs were added using Klenow 59-39 exo-minus, Illumina Solexa sequencing adapters were ligated using T4 DNA ligase (NEB) and amplifi- cation was carried with 10 PCR cycles (15 sec extension time) using Phusion DNA polymerase (Finnzymes) and Illumina Solexa sequencing primers 1.1 and 2.1 (Illumina). Finally, libraries were size selected by electrophoresis and sequenced on an Illumina Genome Analyzer  HiSeq2000.
GEO Accession
GSM1199553