title: GSM4307807: 48hr INCB059872 B H3K4me2; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were lysed by resuspending in cell lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.5% NP-40) and sitting on ice for 10 min. Samples were centrifuged, and nuclei were resuspended in nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 0.5% SDS) and kept on ice for 10 min before sonication using a Bioruptor (Diagenode) on high setting for 15 cycles (30 sec on, 30 sec off). After removing an aliquot to serve as input, chromatin from each sample was divided between two tubes and diluted 1:4 with ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 1.2 mM EDTA pH 8.0, 167 mM NaCl, 1.1% Triton X-100, 0.01% SDS). 3μg anti-H3K4me2 or anti-H3K4me1 was added to each tube. Samples were rotated at 4°C for 2 hours before adding 15μl Protein A Dynabeads and 15μl Protein G Dynabeads. Samples were rotated at 4°C for 1 hour before washing beads 7 times: (twice with low salt buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS), twice with high salt buffer (500 mM NaCl, otherwise same as low salt buffer), once with LiCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 1% NP-40, 1% Na-deoxycholate), and twice with TE. Samples were eluted twice by resuspending beads in elution buffer (10% SDS, 1 M NaHCO3) and rotating for 15 min at room temperature. Libraries were prepared by taking 100 ng DNA from each input and IP sample for polishing, A-tailing, and ligation of adapters. Agencourt AMPure XP Beads were used at 1.8X volume according to manufacturer's instructions for size selection, and eluted material was PCR amplified with Q5 Hot Start DNA Polymerase for 21 cycles. Immediately prior to crosslinking, drosophila S2 cells were added to treated THP-1 cells to serve as spike-in control (~1:27 ratio of drosophila:human cells). Cells were crosslinked with 1% formaldehyde, quenched with glycine, and washed with cold PBS. See "ChIP protocol" column above for experiment-specific protocols.