ChIP-seq analysis of HA-tagged protein

Approximately 10 × 10⁶ DTMYC G401 cells per plate were treated with 500 nM dTAG47 or DMSO for 4 hours and processed for chromatin extraction. Generation of chromatin was performed as described previously (17 (link)). Briefly, following cross-linking the nuclei were extracted and chromatin was fragmented using a Diagenode Bioruptor. Immunoprecipitation was performed on chromatin from 10 × 10⁶ cells using antibodies against HA-epitope (Cell Signaling, C29F4) or normal rabbit IgG control (Cell Signaling, 2729S). Immunoprecipitated DNA was bound to protein A agarose (Roche), washed extensively, and de-crosslinked overnight at 65°C as described previously (17 (link)). Chromatin from three ChIPs were combined and purified using with a PCR purification kit (Qiagen). Eluted DNA was then cleaned-up using AMPure beads and used to create libraries following the Ultra II DNA library Prep protocol with Multiplex Oligos for Illumina (New England BioLabs). Sequencing data were obtained on an Illumina NovaSeq 6000 with 150 bp paired end reads. Sequencing was performed by the Vanderbilt Technologies for Advanced Genomics (VANTAGE) Core at Vanderbilt University Medical Center.