ChIP-Seq Analysis of ASPS Cells

ChIP-Seq was performed using the method previous described with modifications^{58 (link)}. A total of 5 × 10⁶ ASPS cells per immunoprecipitation were cross-linked with 1% formaldehyde for 10 min at room temperature. Chromatin was sheared in SDS lysis buffer containing 1% SDS, 10 mM EDTA, and 50 mM Tris pH 8.0 to an average size of 400 to 500 bp using a Covaris S220 sonicator for 15 min. ChIP was performed with 5 μg anti-histone H3K27ac (Active Motif, 39133), anti-H3K4me3 (Abcam, ab8580), anti-H3K27me3 (Millipore, 07-449), anti-FLAG (Sigma-Aldrich, F7425), anti-ASPSCR1 (Sigma-Aldrich, A026749), anti-BRD4 (Bethyl Laboratories, A301-985A100) antibodies. The antibody-bound protein/DNA complexes were immunoprecipitated using protein G magnetic beads. Immunoprecipitated DNA was then purified and subjected to secondary sonication to an average size 150 to 350 bp. Libraries were prepared according to instructions accompanying the ThruPLEX DNA-Seq kit (Rubicon Genomics). The ChIP DNA was end modified and adapters were ligated. DNA was PCR amplified with Illumina primers and Illumina-compatible indexes were added. The library fragments of approximately 300–500 bp were band-isolated from an agarose gel. The purified DNA was sequenced on an Illumina MiSeq next-generation sequencer following the manufacturer protocols.