ChIP Analysis in Thyroid Cell Lines and Tissues

For ChIP analysis, DTC cells were grown to 85% confluency in 15 cm plates. ChIP was performed as described previously for cell lines^{39 (link)}. For ChIP analysis in normal thyroid tissue, the Novus Biologicals protocol⁴⁰ was followed. Cells were fixed for 8 minutes in 1% formaldehyde. Chromatin was sheared by sonication for 2x [4× 20s on/20s off] by the Bioruptor Pico (Diagenode, Denville, NJ). The following antibodies were used (10 µg per ChIP): anti-CTCF (#2899, Cell Signaling Technology, Danvers, CA), anti-MYC (#9402, Cell Signaling Technology), anti-GABPA (#27795, ThermoFisher), and anti-GSC (#40495, ThermoFisher). DNA was purified by MinElute PCR Purification Kit (Qiagen, Hilden, Germany). ChIP and input DNA were analyzed by qPCR with Sybr Green and melt curve analysis, with the primer sets listed in Supporting Information Table 2. Three replicate PCR reactions were carried out for each sample with two biological replicates, with positive and negative control regions for each antibody. Relative binding was calculated by normalizing the factor binding at the TERT locus relative to input of each sample to the factor binding at the positive control locus.