title: Hi-C of U266-CSCs: DHA
design: U266 CSCs were treated with DHA (5 M) for 48 h, then were harvested and precipitated by centrifugation at 1000 g for 5 min at 4 C. Cells were resuspended in cold PBS and fixed with 1% formaldehyde for 15 min at room temperature. The reaction was quenched by incubating with 125 mM glycine for 5 min at room temperature, then was transferred onto ice. Cells were deposited at 1000 g for 5 min at 4 C, and then washed twice with cold PBS. The cell density was adjusted to 2x106 cells ml-1 and 1 ml was transferred to a 1.5 ml tube. Cells were then pelleted at 1 000 g for 5 min at 4 C. After supernatant aspiration, the pellet was flash frozen in liquid nitrogen and stored at 80 C. Nuclei were isolated with lysis buffer [10 mM Tris-HCl (pH 8.0), 10 mM NaCl and 0.2% IGEPAL CA630], and incubated on ice for 15 min. Pelleted nuclei were permeabilized with 0.5% SDS and incubated for 10 min at 62 C. SDS was quenched by addition of Triton X-100. Two hundred units of DpnII restriction enzyme (NEB, R0147) was used to digest the chromatin. For ligation, 2000U of T4 DNA Ligase (NEB, M0202) was added and samples were incubated at room temperature for 4 h with slow rotation. Hi-C chromatin was sheared to a size around 350 bp using a Covaris instrument (Covaris S2, Woburn, MA). Biotin-tagged DNA was pulled down using Dynabeads MyOne Streptavidin C1 beads (Life technologies, 65002) with 2 Binding Buffer [2 BB: 10 mM Tris-HCl (pH 7.5), 1 nM EDTA, 2 M NaCl]. Each Hi-C library was amplified with 10 cycles of PCR with Illumina primers. Each library was sequenced (150 bp paired-end) using an Illumina platform.