GSM7806678: rep1_SCC25_Fix_FLAG; Homo sapiens; ChIP-Seq
Cells were initially cross-linked with ChIP Cross-link Gold (Diagenode, # C01019027) to be formed reversible DNA-protein links in PBS with 1 mM of MgCl2. After incubation at RT for 30min, the cells were fixed with 11% solution containing formaldehyde (Thermo Fisher #28908), 50mM HEPES-KOH (pH 7.5), 100mM NaCl, 0.5M EDTA, 0.5M EGTA for 10 min. Then, to stop the fixation, 2.5M of Glycine was added and incubated for 5 min. The cells were washed twice with PBS and harvested on ice using cell scrapers. The harvested cells were resuspended in Lysis Buffer 1 (50 mM HEPES-KOH, pH 7.5; 140 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100), allowed to rotate for 10 min at 4°C. After centrifuging, the cells were resuspended in Lysis Buffer 2 (10 mM Tris-HCL pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), allowed to rotate for 10 min at room temperature. After centrifuging, the cells were resuspended in Lysis Buffer 3 (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) and sonicated on a Bioruptor Pico at 4°C for 20 min with sonication beads. The sonicated cells were centrifuged, and the supernatant was transferred to new tubes.  5 μg of antibodies were used along with 2 μg of Spike-in antibodies (Active Motif, #61686) were bound to 50 μl of Dynabeads Protein G (Thermo Fisher, #10004D), previously washed with 0.5% BSA in PBS and rotated end-to-end overnight at 4°C. 30 μg of shared chromatin and 20 ng of spike-in chromatin from Drosophila (Active Motif, #53083) were incubated with antibodies/beads mixture overnight at 4°C. The beads were washed 6 times with Wash Buffer (50 mM Hepes-KOH, pH 7.6; 500 mM LiCl, 1 mM EDTA, 1% NP-40; 0.5% Na-Deoxycholate) and once with TE containing 50 mM NaCl. After elution of immunocomplexes from beads with Elution Buffer (50 mM Tris-HCl, pH 8; 10 mM EDTA, 1% SDS) by incubation at 65°C for 15 min, the supernatant was reverse crosslinked at 65°C for 3hrs, treated sequentially with RNAse A (Sigma, #R5503) at 37°C for 2hrs and then treated with Proteinase K (NEB, #P8107S) at 55°C for 2 hrs. DNA purification was performed with the QIAquick PCR Purification Kit (Qiagen, # 28106). Immunoprecipitated DNA was used to generate libraries using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs cat# 7370) following the manufacturer's instructions.