GSM2610546: LNCaP DNase-seq; Homo sapiens; DNase-Hypersensitivity
Cells (7x10^6 per sample) were scraped, centrifuged and washed with PBS. Cell pellets were resuspended in nuclear extraction buffer (10mM Tris-HCl pH7.4, 12.5mM NaCl, 3mM MgCl, 0.1mM EDTA, 0.5% IGEPAL) and dounced until nuclei were visible under light microscope with 0.4% Trypan Blue staining. DNase1 (Roche, #04716728001) was added to nuclei pellets of LNCaP (24U) and PrEC (12U) and incubated at 37°C for 15 minutes. DNaseI reactions were terminated by the addition of 36mM EDTA and Proteinase K was added before incubating at 55°C overnight. DNA was purified by phenol-chloroform extraction and ethanol precipitation. Samples were separated using electrophoresis on a 1% agarose gel. 100-300bp sections were excised and purified using the QIAquick Gel Extraction kit. Library preparation and sequencing were performed by the USC Epigenome Center. Libraries were prepared using the Illumina TruSeq Chip Library Prep Kit. The resulting libraries were sequenced on the Illumina HiSeq 2000 or 2500 platform configured for 50bp single-end reads.