title: GSM2110110: MEL cells 1% 10 min fixation; Mus musculus; OTHER
library_construction_protocol: 7.5 million cells were fixed for 10 or 15 minutes by resuspending cell pellet in 7.5 ml full culture medium supplemented with 1% or 2% formaldehyde (Sigma), respectively. The reaction was quenched by addition of 2M glycine to a final concentration of 0.2 M and incubation for 5 minutes at room temperature on a rocker. Cells were pelleted by centrifugation at 300 g for 5 minutes at 4°C, washed by resuspension in 1 ml of PBS, pelleted and resuspended in 1 ml of PBS again. 1.9 mln cells from each sample were taken, the rest were snap-frozen for other experiments. The samples were pelleted again, then resuspended in 300 μl of cold lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% NP-40 substitute (Sigma), 1× Halt™ Protease Inhibitor Cocktail (Thermo Scientific)) and incubated on ice for at least 15 minutes. Cells were pelleted by centrifugation at 2,500g for 5 minutes, resuspended in 500 µl of lysis buffer and pelleted again. The pellet was resuspended in 50 μl 0.3% SDS in NEBuffer 3 and incubated at 62°C for 10 minutes. Then the suspension was diluted with 145 μl of 1× NEBuffer 3, 12.5 μl mQ and 12.5 μl of 20% Triton X-100 and incubated at 37°C for 1 hour to quench SDS. 30 µl were taken as a chromatin integrity control. The cells were pelleted by centrifugation at 3,000g for 5 minutes, resuspended in 250 μl of 1× DpnII buffer (NEB) with 600 U DpnII enzyme (NEB), and the chromatin was digested overnight (14-16 hours) at 37°C with rotation. In the morning 200 more units were added and the cells were incubated for additional 2 hours. DpnII was inactivated by incubation at 65°C for 20 minutes. 40 μl was taken as a digestion control, 40 μl of mQ were added to the sample. The ends were filled-in by addition of 50 μl fill-in mix (36 ul mQ, 1.5 μl of 10 mM biotin-14-CTP, 1.5 μl of 10 mM dATP, 1.5 μl of 10 mM dGTP, 1.5 μl of 10 mM dTTP, 8 μl of 5 U/μl DNA Polymerase I, Large (Klenow) Fragment (NEB)) and incubation at 37°C for 1.5 hours. Then 900 μl of ligation mix (712 μl water, 120 μl 10× NEB T4 DNA ligase buffer (NEB), 50 μl 20% Triton X-100, 12 μl 10 mg/ml BSA (NEB), 6 μl 5 U/μl T4 DNA Ligase (Thermo Scientific)) were added to the sample. The sample was mixed by inversion and incubated with rotation for 4 hours at room temperature. Then the nuclei were pelleted by centrifugation at 3,000g for 10 minutes and resuspended in 200 μl mQ. Proteins were degraded by addition of 15 μl of 20 mg/ml proteinase K and 20 μl of 10% SDS and incubation at 55°C for 30 minutes. Then 130 μl 5M NaCl were added and the samples were incubated at 65°C overnight (14-16 hours). The DNA was ethanol precipitated and then dissolved in 500 μl 10 mM Tris-HCl pH 8.0, washed twice on Amicon 30K filter units (Millipore) with 10 mM Tris-HCl pH 8.0 and then eluted into a clean tube. The volume was adjusted to 500 μl with sonication buffer (50 mM Tris, 10 mM EDTA, 0.1% SDS) and the samples were sonicated using VirSonic 100 (Virtis) with power setting 15 for 15 seconds twice with 1 minute interval. Samples were washed twice again using Amicon 30K filter units (Millipore). All consecutive steps until PCR were performed in LoBind tubes (Eppendorf). 30 µl of 10 mg/ml Dynabeads MyOne Streptavidin T1 beads (Life technologies) were prepared for biotin pull-down by washing with 80 µl of 1× TWB (1× Tween Washing Buffer, 5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween 20). Then the beads were resuspended in 60 μl of 2× BB (2× Binding Buffer: 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl) and added to the samples; the suspension was incubated for 15 minutes at room temperature with rotation. After that the beads were used for library preparation. 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: GSM2110110