CUT&RUN for Transcription Factor Mapping

CUT&RUN was performed as previously described (13 (link)). Briefly, Nthy-ORI cells were harvested after 6 h of treatment with T₃ or vehicle control, washed, and bound to activated Concanavalin A coated magnetic beads (Epicypher 21-1401). Cells were then permeabilized with Wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine 0.05% digitonin). Permeabilized cells were then incubated with the indicated antibody (Supplemental Table S3) at 4°C with constant agitation overnight. Cells were washed twice more before incubation with recombinant p-AG MNase (Epicypher 15-1016) at 4°C for 2 h. Liberated DNA was purified, and libraries were prepared using the NEB Ultra FS II DNA Library Kit (NEB E6177) and amplified with 14 cycles of PCR. Amplified libraries were then purified with AMPure beads (Agencourt), quantified via Qubit (Life Technologies), and quality was assessed using the BioAnalyzer (Agilent) High-Sensitivity DNA kit. CUT&RUN libraries were pooled and sequenced on the Illumina HiSeq 1500 platform with 100 bp paired-end reads.