Dialyzed fetal bovine serum

MCF-7 cells were grown in a label-free DMEM formulation and treated with 200 µM 4-thiouridine (4sU, ChemGenes) for 16 h, followed by IR exposure (10 Gy) or no treatment. One hour after IR exposure, the cells were UV crosslinked (365 nm, 0.2 J/cm²) and lysed in lysis/binding buffer (Baltz et al. 2012 (link)). To obtain SILAC “heavy” labeled cell extracts, HEK293 Flp-In T-REx cells were grown in high glucose SILAC DMEM (PAA, E15-086) supplemented with 10% (v/v) dialyzed fetal bovine serum (Sigma Aldrich), 2 mM L-glutamine, 100 U/mL penicillin/streptomycin (both from Thermo Fisher Scientific), 0.398 mM ¹³C₆,¹⁵N₂ L-arginine, and 0.798 mM ¹³C₆,¹⁵N₂ L-lysine (Cambridge Isotope Laboratories) for at least seven passages. Cells were then 4sU-treated and crosslinked as described above. Cell lysates prepared from MCF-7 label-free cells were mixed with equal volumes of SILAC “heavy” HEK293 lysates, and oligo(dT) affinity purification was performed as previously described (Baltz et al. 2012 (link)). Details are provided in Supplemental Methods. Protein–RNA complexes were heat-eluted at 80°C in 10 mM Tris[pH 7.5]. RNA was removed by treatment with RNase I (Thermo Fisher Scientific) and benzonase (Merck), and proteins were concentrated with an Amicon filter device (Millipore UFC901024).