title: GSM2109886: K562_bulkB; Homo sapiens; OTHER
library_construction_protocol: 2-4 million cells were fixed for 10 minutes by resuspending the cell pellet in 5 ml full culture medium supplemented with 1% formaldehyde (Sigma). The reaction was quenched by addition of 2 M glycine to a final concentration of 125 mM and incubation for 5 minutes on ice. Cells were pelleted by centrifugation, washed by resuspension in 1 ml of PBS and pelleted again. Then the cells were resuspended in 1.5 ml of lysis buffer and incubated on ice for at least 15 minutes. Cells were pelleted by centrifugation at 2,500g for 5 minutes, resuspended in 100 μl of 1×NEBuffer 3 and pelleted again. The pellet was resuspended in 100 μl of 0.3% SDS in 1×NEBuffer 3 and incubated at 37°C for 1 hour. Then the suspension was diluted with 330 μl of 1× NEBuffer 3 and 53 μl of 20% Triton X-100 (Sigma), and incubated at 37°C for 1 hour to quench SDS. The cells were pelleted by centrifugation at 3,000g for 5 minutes, resuspended in 250 μl of 1× DpnII buffer (NEB). 20 μl were taken as a chromatin integrity control, then 600 U DpnII enzyme (NEB) were added, and the chromatin was digested overnight at 37°C with rotation. In the morning 200 more units of DpnII were added and the cells were incubated for additional 2 hours. DpnII was inactivated by incubation at 65°C for 20 minutes. 20 μl was taken as a digestion control. Nuclei were pelleted by centrifugation at 3,000g for 5 minutes, resuspended in 100 μl of 1× T4 DNA ligase buffer (Thermo Scientific) and pelleted again. The pellet was resuspended in 500 μl of 1× T4 DNA ligase buffer, and 50 U T4 DNA ligase (Thermo Scientific) were added. The sample was mixed by inversion and incubated with rotation for 4 hours at 16°C and then for 30 minutes at room temperature. Then the nuclei were pelleted by centrifugation at 3,000g for 5 minutes and resuspended in 120 μl of sterile PBS. 20 μl were taken as a ligation control and another 20 ul were used to for DNA purification, library preparation and sequencing. The library preparation was done by the VBCF NGS unit (vbcf.ac.at) following the standard DNA sample  preparation protocol for Solexa platforms (NEB reagents).
attributes.tag: GEO Accession
attributes.value: GSM2109886