title: GSM5655011: HeLa-WT-DNMT1-ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were crosslinked using ChIP-gold crosslinking reagent (Diagenode) according to manufacturer's instructions followed by DNA-protein crosslinking with formaldehyde. Formaldehyde fixation was done by adding (1/100 of the volume of the plate) 11% solution containing formaldehyde (Thermo Fisher), 50mM HEPES-KOH (pH 7.5), 100mM NaCl, 0.5M EDTA, 0.5M EGTA for 10 min. 1/20 of the plates volume equivalent of freshly prepared Glycine 2.5M was added to quench the reaction. After 5 min, supernatant was aspirated, and the cells were washed twice with 5 ml 1xPBS and harvested on ice using cell scrapers. Cells were pelleted at 2950 x g for 10 min at 4°C, washed with 10 ml cold 1xPBS twice. Cell pellets were flash freeze in liquid nitrogen and store at -80°C. To perform chromatin immunoprecipitation, the cell pellets collected from 5 plates of cells were resuspended in 5 ml of 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, and pelleted at 2950 x g for 10 min at 4°C. Cells were then resuspended in 5 ml of 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, and pelleted at 2,950 x g for 10 min at 4°C. Cells were then resuspended in 3 ml of 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 then transfer to 15 ml sonication tubes (Diagenode) containing 1.2g of sonication beads. Cells were sonicated on a Bioruptor Pico at 4°C for 20 min of 30'' ON-OFF cycles. After sonication, samples were centrifuged at 20,000 x g for 15 min at 4°C and supernatant was transferred to new 15 ml tubes. To quantify and check sonication efficiency, 50 µl of sonicated samples was transferred to a new microtube with 50µl PBS and incubated at 65°C for 3 h on an Eppendorf Thermomixer shaking at 1000 rpm to de-crosslink. DNA was purified with the QIAquick PCR Purification Kit (Qiagen) and eluted in 50 µl of H20. 1μg of de-crosslinked chromatin from each sample were run in a 1% agarose gel at 100V for 45 min and imaged on a Bio-rad ChemiDoc XRS+. DNA concentration was measured by Qubit. 10 µg of DNMT1 antibody (Active Motif) were bound to 50 µl of Dynabeads Protein G (Thermo Fisher), previously washed with 1 ml of 0.5% BSA in 1x PBS three times and rotated end-to-end overnight at 4°C (Eppendorf). Next morning, beads were washed with 1 ml of 0.5% BSA in 1xPBS three times. 30 g of chromatin was added to 1.5 ml LoBind tube (Eppendorf) containing pre-bound antibodies beads and brought up to 500 µl final volume with Lysis Buffer 3 and rotated end-to-end overnight at 4°C. 5 µl was removed as input material (1%) and placed in a separate microtube at 4°C completing the volume to 50 L with Lysis Buffer 3. Beads were washed 6 times with 1 ml 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 1 ml TE containing 50 mM NaCl. Beads were dried after the last wash by removing the TE buffer. 210 l of Elution Buffer (50 mM Tris-HCl, pH 8; 10 mM EDTA, 1% SDS) was added to each sample and 150 µl elution buffer were added to each input sample that was previously set aside. Immunocomplexes were eluted from beads by incubation at 65°C for 15 min on an Eppendorf Thermomixer shaking at 1000 rpm. Tubes were centrifuged for 3 min at 960 g at RT and 200 µl of supernatant was transferred to a new tube and reverse crosslinked overnight at 65°C. Next day, 200 l of TE was added to each de-crosslinked sample and input, and the samples were treated sequentially with 0.2 mg/ml RNAse A (Sigma) at 37°C and 0.2 µg/ml Proteinase K (NEB) at 55°C for two hours each. DNA purification was performed with the QIAquick PCR Purification Kit (Qiagen) and eluted in 50 µl of H2O and quantified by Qubit. Immunoprecipitated DNA was used to generate libraries using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs) following the manufacturer's instructions.