GSM1252095: nucleosome J1; Mus musculus; MNase-Seq
Collect 10ml cells in a 15ml BD tube, and fix in 1% formaldehyde for 3 minutes at room temperature. Formaldehyde was quenched by adding 1ml 1.24M glycine. Cells were rinsed 3 times in ice-cold PBS, immediately resuspended in cell lysis buffer (10mM Tris-HCl pH7.5/10mM NaCl/0.5%NP40) and lysed for 3 to 4 min on ice. Nuclei were collected by centrifugation, washed with ice-cold PBS, collected by centrifugation again and resuspended in digestion buffer (50mM Tris-HCl pH7.5/1mM CaCl2/0.2%Triton X-100). Samples were divided into 200μl aliquots, prewarmed to 25°C and incubated with MNase (stored in MNase storage buffer (10mM Hepes pH7.5/100mM NaCl/1mM CaCl2/50% glycerol) and diluted in MNase dilution buffer (50mM Tris-HCl pH8/10mM NaCl/126mM CaCl2/5% glycerol)). Concentration of MNase and time of incubation were optimized to obtain 80% mononucleosomes as determined by BioAnalyzer analysis. Libraries were prepared using the Illumina sequencing library preparation protocol.
GEO Accession
GSM1252095