ChIP-Seq was carried out using the method previously described using a biological duplicate (58 (link)). Briefly, 5 × 106 mesenchymal chondrosarcoma cells per immunoprecipitation were cross-linked with 1% formaldehyde for 10 minutes 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 400–500 bp using a Covaris S220 sonicator for 15 minutes. ChIP was carried out with 5 μg anti-FLAG (Sigma-Aldrich), anti-histone H3K27ac (Active Motif), anti-histone H3K27Me3 (Abcam), anti-histone H3K4Me3 (Abcam), anti-Runx2 (Cell Signaling), or anti-Runx3 (Cell Signaling) antibodies. The antibody-bound protein/DNA complexes were immunoprecipitated using ChIP grade protein G magnetic beads (Cell Signaling).
Immunoprecipitated DNA was then purified and subjected to secondary sonication to an average size of 150–350 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.
Immunoprecipitated DNA was then purified and subjected to secondary sonication to an average size of 150–350 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.