GSM4498668: 1-Rep2-input; Homo sapiens; OTHER
COSMIC was performed as previously described (Erwin,G.S., Bhimsaria,D., Eguchi,A. and Ansari,A.Z. (2014) Mapping Polyamide–DNA Interactions in Human Cells Reveals a New Design Strategy for Effective Targeting of Genomic Sites. Angew. Chemie Int. Ed., 53, 10124.).  Briefly, cells or nuclei were centrifuged at 500 × g and re-suspended in COSMIC buffer [20 mM Tris·Cl (pH 8.1), 2 mM EDTA, 150 mM NaCl, 1mM PMSF, 1mM benzamidine, 1.5 μM pepstatin,1% Triton X-100, 0.1% SDS]. Samples were sonicated at 3 °C for 36 min with a cycle of 10 s ON and 10 s OFF, at HIGH setting (Bioruptor Plus, Diagenode). Samples were centrifuged 10 min at 12,000 × g and 10% of the sample was saved as input DNA and stored at -80 °C until reversal of cross-linking.  The rest of the sample was used for the affinity purification. Streptavidin-coated magnetic beads (100 μL per sample, Dynabeads MyOne C1) were washed in COSMIC buffer and incubated with samples for 16 h at 4°C.  All washes were performed at room temperature unless otherwise noted. For 1 and 2, 1A and 2A were added (5 μM), respectively, in the washes. Samples were washed twice with COSMIC buffer (once 12 h and once 4 h). Samples were then washed once with washing buffer 1 [10 mM Tris·Cl (pH 8.0), 1 mM EDTA, 3% (v/v) SDS], once with washing buffer 2 [10 mM Tris·Cl (pH 8.0), 250 mM LiCl, 1 mM EDTA, 0.5% Nonidet P-40, 1% sodium deoxycholate], twice with freshly prepared washing buffer 3 [4 M urea, 10 mM Tris·Cl (pH 7.5) at 50 °C, 1 mM EDTA, 0.1% Nonidet P-40], and twice with TE buffer [10 mM Tris·Cl (pH 8.0), 1 mM EDTA]. Samples were re-suspended in TE and labelled as AP DNA.  Input and AP samples were re-suspended in cross-link reversal buffer [10 mM Tris (pH 7.6), 0.4 mM EDTA, 100 mM KOH]. Crosslinks were reversed, and DNA was eluted from beads at the same time by heating samples for 30 min at 90 °C. Input and AP samples were neutralized with 6N HCl, and incubated first with RNase A (0.2 μg/μL) for 1 h at 37 °C and then with Proteinase K (0.2 μg/μL) for 1 h at 55 °C. Samples were purified with the MinElute PCR Purification Kit (Qiagen). Sequencing libraries for Input and AP DNA were labeled with a DNA barcode for multiplexing, and prepared for sequencing with the TruSeq ChIP Sample Prep Kit (Illumina, Inc., San Diego, CA) per manufacturer's directions.