GSM5709519: Differentiating, low glucose, DDX21 epitope, donor 2, ChIP-seq; Homo sapiens; ChIP-Seq
Cells were lysed with 500 µL swelling buffer (100 mM Tris-HCl pH 7.5, 10 mM KOAc, 15 mM MgOAc, 1% NP-40 and 1X protease inhibitor, EDTA-free) on ice for 10 minutes and transfered into a pre-chilled 2 ml Dounce homogenizer (Type B) to break cells open using 10 strokes of a tight pestle. After centrifugation for 5 minutes at 4000 rpm, 4℃, nuclei were lysed with 10X the amount of nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA and 1X protease inhibitor, EDTA-free) on ice for 10 minutes, transfered to Polystyrene tubes and sonicated to shear the chromatin to 150-250bp (Bioruptor UCD-200). 40-100 µg sheared chromatin was input to either DDX21- or HA-immunopurification. Sheared chromatin was diluted 5-8-fold in ChIP dilution buffer (16.7 mM Tris-HCl, pH = 7.5, 167 mM NaCl, 1.2 mM EDTA, 1.1% TritonX-100, 0.01% SDS). 1 µg DDX21 antibody (Novus Biologicals, NBP1-83310) was added for each 20-µg chromatin and incubated overnight at 4℃. 60 µL protein G dynabeads (Invitrogen, 10004D) was used for each 50-µg sheared chromatin, and incubated for 2 hours at 4℃ followed by washing with buffer A (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100, 0.1% SDS, 0.1% NaDOC), buffer B (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% TritonX-100, 0.1% SDS, 0.1% NaDOC), buffer C (50 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% NaDOC) and finally buffer D (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), each wash performed twice. Beads were then incubated at 65℃ with elution buffer (10 mM Tris-HCl pH 8.0, 300 mM NaCl, 5 mM EDTA, 0.5% SDS) for 15 minutes. Eluted DNA was incubated at 67℃ overnight to reverse crosslinks, followed by adding 1 µL 10 mg/mL RNaseA (Qiagen, 1032724) and incubating at 37℃ for 30 minutes to remove RNA. The resulting DNA was further purified using a PCR purification kit (Qiagen, 28106) and quantified by Qubit High Sensitivity dsDNA kit (Invitrogen, Q32851). One µg of input DNA and ChIP DNA were input to ChIP library preparation using the NEBNext Ultra II DNA library prep kit (NEB, E7103S), following the manufacturer's protocol.