Name: GSM6412298
Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Cells for ChIP were cultured in 15 cm plates (~10 million cells). Cell pellets were first washed with cold PBS, crosslinked at room temperature with 1% formaldehyde (ThermoFisher Scientific) for 8 min. Crosslinking reactions were quenched by addition of 125 mM glycine for 10 min. Cell were then resuspended in Swelling Buffer (25 mM Hepes pH 7.8, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT) followed by Dounce homogenization. Nuclei were pelleted by centrifugation and then resuspended in sonication buffer (0.1% SDS, 1 mM EDTA and 10 mM Tri-HCl pH 8.0). The nuclei were sonicated to shear chromatin into ∼200-500 bp fragments using a Covaris E220. Sonicated samples were diluted with ChIP dilution buffer (0.1% SDS, 1 mM EDTA and 10 mM Tri-HCl pH 8.0, 1% Triton X-100, 150 mM NaCl). Diluted samples were centrifuged at 13,000 rpm for 10 min. The supernatant was used for immunoprecipitation using antibodies and 25 μl protein A/G beads for 12-16 h at 4°C (see Table S9 for antibodies). For H3K27me3 ChIP-Seq, Drosophila S2 chromatin (Active Motif# 53083) and histone H2Av antibody (Active Motif# 61686) were added as spike-in controls. ChIP-Seq samples for Flag antibody do not have spike-in controls. The beads were washed twice with high salt wash buffer A (50 mM Hepes pH 7.9, 500 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% Sodium deoxycholate, and 0.1% SDS), twice with wash buffer B (20 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% Sodium deoxycholate, 0.5% NP-40) and twice with 1X TE (10 mM Tris-HCl, 1mM EDTA). The bound chromatin fragments were eluted with elution buffer (50 mM Tris pH 8.0, 1 mM EDTA, 50 mM NaHCO3,1% SDS) twice for 10 min each at 65°C. Eluted DNA-proteins complexes were incubated overnight at 65°C to reverse crosslinks. RNAase A followed by Proteinase K was then added to digest RNA and protein. DNA was further purified using phenol chloroform/PCR Purification Kit (QIAGEN) For ChIP-seq, sequencing library was constructed using TruSeq DNA sample Prep Kits (Illumina) and adapter dimers were removed by 2% agarose and Tris-acetate-EDTA gel electrophoresis. Size selected and purified DNA libraries were sequenced on an Illumina NextSeq 500 machine (Bauer core facility at Harvard University) to obtain 75 bp single-end reads.