SLAM-seq

SLAM-sequencing protocols were followed as previously described.58 In brief, either parental CH22 and HA-dTAG-T, T^−/− CH22 chordoma cells were seeded the night before addition of compound in 6-cm tissue culture-treated dishes at 750,000 cells per dish. The following morning, media was replaced to contain DMSO or THZ1 (60nM) or degron at (1μM). One hour later, media was replaced with DMSO or compound containing media and s⁴U (Sigma T4509) to a final concentration of 750 μM. Media containing compound and s⁴U were exchanged every 3 hours for the duration of the pulse, before collecting the cells at 8 hours post initial addition of compound. Cells were harvested by total RNA extraction using TRIzol (with 0.1mM DTT during isopropanol precipitation). RNA was resuspended in 1mM DTT and ERCC RNA Spike-In Control Mixes (ThermoFisher 4456740) were added to each sample to allow for cell count normalization of gene expression. 5μg of total RNA was treated with 10mM iodoacetamide (Sigma I1149) under denaturing conditions, ethanol precipitated and purified, and subjected to Quant-seq 3′ end mRNA library preparation for Illumina (Lexogen 015). Libraries were sequenced using the Illumina Nextseq 500 instrument with single-end, 75bp reads.