GSM8799006: MDAMB468_H4K20me3_Vehicle_rep1; Homo sapiens; ChIP-Seq
For ChIP-seq, 150 mm plates tissue culture plates were fixed in 1% formaldehyde (Electron Microscopy Sciences, 15714) containing fixing buffer (50 mM HEPESKOH pH7.5, 100 mM NaCl, 1 mM EDTA pH8 and 0.5 mM EGTA pH8) for 10 min at 37°C.  Crosslinking was quenched by adding glycine to a final concentration of 0.125 M and further rocking the plates for 5 min. Cell monolayers were washed twice with ice-cold PBS and scrapped off the plate in 10 ml PBS, spun at 200g for 5 min, and flash frozen after removing the supernatant. Frozen pellets were resuspended in 1 ml of Cell Lysis Buffer (50 mM HEPES pH8, 140 mM NaCl, 1 mM EDTA pH8, 10 % glycerol, 0.5% NP-40, 0.25% Triton X-100 and PPI) and incubated for 10 min at 4°C with gentle rotation. Nuclei were then centrifuged at 1,700g for 5 min at 4°C. Pelleted nuclei were washed twice with 1 ml Wash Buffer (10 mM Tris-HCl pH8, 200 mM NaCl, 1mM EDTA pH8, 0.5 mM EGTA pH8 and PPI) and then resuspended in 1 ml of Shearing Buffer (10 mM Tris-HCl pH7.4, 1 mM EDTA pH8, 0.1% SDS, 1% Triton-X100, 0.1% Sodium Deoxycholate, 0.25% NLauroylsarcosine, 1 mM DTT and PPI) and sonicated in a Covaris E220 focused-ultrasonicator (peak intensity: 140, 5% duty cycle, 200 cycles per burst, 10-12 min) using 1 ml AFA fiber tubes. An equivalent of 5 x 106 cells was sonicated in each tube and used for each ChIP. Lysates were centrifuged for 15 min at 10,000g in a refrigerated centrifuge. Cleared supernatants were transferred to new tubes and NaCl was added to a final concentration of 150 mM. Samples were then incubated with Dynabeads Protein G (LifeTechnologies,10003D, 40 µl/ml of lysate) for 1 hour at 4°C, with constant rotation (pre-clearing). After magnetic separation of the beads, the appropriate primary antibody was added to each tube for an overnight immunoprecipitation, at 4°C, with gentle rotation. Antibody-bound cross-linked chromatin was precipitated with prewashed Dynabeads Protein G for 2 h at 4°C and with constant rotation. Beads were then washed sequentially with low salt wash buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 10 mM EDTA, and 1% SDS), high salt wash buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, and 1% SDS), LiCl wash buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, and 1% SDS) and twice with 1X TE buffer. Each wash was performed for 5 min at 4°C with gentle rotation. DNA was eluted from the beads with 300 µl of 100 mM sodium bicarbonate and 1% SDS for 30 min at RT with constant shaking. Crosslinking was reversed overnight at 65°C and RNA and protein were then sequentially digested by adding 0.2 mg/ml RNase A for 30 min at 37°C followed by 0.2 mg/ml Proteinase K for 1 h at 55°C. DNA was extracted with 300 µl of phenol/chloroform/isoamyl-alcohol (Calbiochem, #516726) and centrifuged at 20,000g for 5 min at RT. DNA in the upper layer was then precipitated with 1 volume isopropanol, 1/3 volume of 2 M sodium perchlorate and 5 µl glycogen and centrifuged at 20,000g for 10 min at RT. Pelleted DNA was washed twice with 70% ethanol and resuspended in 20 µl low TE. For ATAC-seq: Fifty thousand cells were resuspended in 1ml of cold ATAC-seq resuspension buffer (RSB) (10mM Tris-HCl (pH 7.4), 10mM NaCl and 3mM MgCl2 in water). Cells were centrifuged at maximum speed for 10min in a prechilled (4 °C) fixed-angle centrifuge. After centrifugation, supernatant was carefully aspirated. Cell pellets were then resuspended in 50μl of ATAC-seq RSB containing 0.1% NP40, 0.1% Tween-20 and 0.01% digitonin by pipetting up and down three times and incubated on ice for 3min. After lysis, 1ml of ATAC-seq RSB containing only 0.1% Tween-20 was added and the tubes were inverted to mix. Nuclei were then centrifuged for 5min at max speed in a prechilled fixed-angle centrifuge. Supernatant was removed and nuclei were resuspended in 50μl of transposition mix (25μl 2X TD buffer, 2.5μl transposase (100nM final), 16.5μl PBS, 0.5μl 1% digitonin, 0.5μl 10% Tween20 and 5μl water46) by pipetting up and down six times. Transposition reactions were incubated at 37 °C for 30min in a thermomixer with shaking at 1,000 r.p.m. Reactions were cleaned up with Qiagen MinElute columns. ChIP-seq/Hi-ChIP: Libraries were prepared using the ThruPLEX DNA-seq Kit (Rubicon Genomics, R400427) according to the manufacturer's protocol. Library fragments were size-selected by PAGE within a 150kb and 700kb range. ATAC-seq: Libraries were amplified as previously described (Buenrostro et al., 2015).