ChIP-Seq Analysis of (R)-9b Effects

Cell pellets were resuspended in RLB buffer and sonicated and the soluble chromatin was incubated with antibodies and Protein-G/A magnetic beads. The complexes were washed with RLB buffer, followed by ChIP buffer 1 and 2 (Active Motif), eluted with elution buffer and subjected to Proteinase-K treatment. ChIP DNA was purified using PCR DNA purification columns (Qiagen). For ChIP-Seq, MDA-MB-453 cells (5 × 10⁷ cells) were treated with vehicle or (R)-9b. Cell pellets were resuspended in RLB buffer and sonicated for 25 s. The soluble chromatin was incubated overnight at 4 °C with antibodies and protein-G/A magnetic beads [21 (link)]. Ten nanograms of immunoprecipitated DNA was used to generate sequencing libraries using the Kapa Hyper Prep Kit (Roche Sequencing Solutions Inc., Pleasanton, CA). The size and quality of the library was evaluated using the Agilent BioAnalyzer (Agilent Technologies, Inc., Santa Clara, CA), and the library was quantitated with the Kapa Library Quantification Kit. Each enriched DNA library was then sequenced on an Illumina NextSeq 500 sequencer to generate 40–50 million 75-base paired-end reads (Illumina, Inc., San Diego, CA). The raw sequence data were aligned using BowTie 2 [68 (link)], and the binding sites were identified using the MACS peak-finding software [69 (link)].