ChIP-seq protocol for transcription factors

ChIP-seq was performed as previously described (30 (link)). Briefly, cells were cross-linked with 1% paraformaldehyde for 10 min and were quenched with glycine for 5 min. After cell lysis, fixed chromatin was fragmented to 200- to 600-bp DNA size using a Diagenode Bioruptor Plus. DNA fragments were immunoprecipitated with indicated antibodies and Protein A/G beads (Smart-Lifesciences, #SA032100). Specific antibodies against MYCN, MAX, AFF1, and AFF4 were generated by immunization in rabbits with recombinant antigens. One to 10 ng of immunoprecipitated DNA was used for DNA library preparation. ChIP-seq raw reads were aligned to the human genome (hg38) with Bowtie (version 1.1.2) using default settings. Read coverage was performed with deepTools and visualized with the UCSC genome browser. Peaks were called using MACS2 (version 2.1.2) with default parameters and a P value cutoff at 1 × 10⁻⁵ before annotation by ChIPseeker (version 1.28.3). Heatmaps and metagene plots were generated using the computeMatrix command from deepTools and NGStools, respectively.