GSM6260193: MDA-MB-231 sgNT1, input, biological replicate1; Homo sapiens; ChIP-Seq
Around 2 x 107 MLL3-WT or -mutant MCF7 and MDA-MB-231 cells were cross-linked with 1% formaldehyde for 10min, quenched by 125 mM glycine for 10 min and rinsed twice with 1X PBS. Fixed cells were incubated in the lysis buffer containing 5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40 and protease inhibitors on ice for 10 min and centrifuged at 3000 rpm for 5min at 4°C. Nuclear pellets were resuspended with cold TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA), and sonicated into 200-500 bp fragments at 4°C. The experimental chromatins mixed with 20 ng drosophila spike-in chromatin (Active Motif, #53083) were incubated overnight at 4°C with Dynabeads Protein A (Invitrogen, 10002D), which had been pre-coated with 1 µg Drosophila-specific histone variant H2Av spike-in antibody (Active Motif, #61686) and 8 µg primary target antibody. The beads were washed twice with RIPA buffer, twice with RIPA buffer containing 300 mM NaCl, twice with LiCl buffer (50 mM Tris-Cl pH 7.5/250 mM LiCl/0.5% NP-40/0.5% Na-DOC) and twice with PBS. DNA was then eluted with 100 μl elution buffer (0.1 M NaHCO3, 1%SDS and 20 μg proteinase K) and reverse crosslinked at 65°C overnight. DNA was purified using QIAquick PCR purification kit (Qiagen) and quantified. The ChIP DNA and input DNA were used to construct libraries using NEBNext Ultra™ II DNA Library Prep kit for Illumina (NEB, E7645) following the manufacturer's instructions. The final libraries were sequenced on Illumina NovaSeq 6000.