title: GSM2066271: human_mono.bam; Homo sapiens; MNase-Seq
library_construction_protocol: Following liquefaction, sperm were washed twice and frozen as dry pellets in liquid nitrogen. Sperm were thawed on ice and washed twice with PBS. Following filtration through an 80-micron mesh, the cells were washed twice and resuspended in 1 mL of a PBS solution containing 0.5% Triton X-100. The absence of somatic cells was confirmed by light microscopy. The cells were incubated for 10 minutes on ice with occasional mixing. Subsequently, ice-cold PBS was added to 10 mL and the cells were washed twice prior to counting with a hemocytomoter. Tritonized sperm suspensions were adjusted to 5x10^6 cells/mL in 5 mM CaCl2, 10 mM dithiothreitol, buffered with 50 mM Tris-HCl, pH 7.9. The suspension was placed 37°C for 30 minutes. Subsequently, the sample was diluted with prewarmed reaction buffer to a final concentration of 5x10^6 cells/mL in 5 mM CaCl2, 10 mM dithiothreitol, 2 Kunitz unit/mL MNase (New England BioLabs), buffered with 50 mM Tris-HCl, pH 7.9. The sample was digested for 5 minutes and the reactions were stopped with the addition of 0.5 M EDTA pH 8.0 to a final concentration of 20 mM. The cell suspension was then rotated at 4°C for 30 minutes prior to centrifugation at 20,000 rcf for 10 minutes. Soluble nucleosome-bound DNAs were recovered from the supernatant by proteinase K digestion and subsequent phenol-chloroform extraction and ethanol precipitation. Purified DNAs were resolved by gel electrophoresis. Bands corresponding to the length of DNA associated with a mononucleosome (~150 bp) were excised and column purified. DNAs were used to construct multiplexed DNA sequencing libraries with the NEBNext ChIP-Seq kit (New England BioLabs). All libraries were subjected to 50 cycles of paired-end sequencing on the Illumina HiSeq 2500 platform.
attributes.tag: GEO Accession
attributes.value: GSM2066271