Cells were washed once with phosphate-buffered saline (PBS) before ultraviolet cross-linking at 900 mJ/cm2 using a Stratalinker (Stratagene). RIP lysis buffer [50 mM tris-HCl (pH 8.0), 150 mM NaCl, 1 mM MgCl2, 10% (v/v) glycerol, 1% (v/v) NP-40, 1 mM dithiothreitol, 0.2 mM sodium orthovanadate, and protease inhibitor cocktails] was added directly to the plate, on ice. The lysate was agitated at 4°C for 10 min before sample clarification at 13,000 rpm. For protein samples, 5% of inputs were taken and boiled in an SDS-loading buffer. For RNA samples, 10% of inputs were taken, and 10 μg of proteinase K was added for 30 min at 37°C before addition of TRIzol and RNA isolation. The rest of the lysate was precleared using preblocked protein A/G agarose beads, 1 μg of nonspecific IgG (Jackson ImmunoResearch), and heparin (0.1 mg/ml) for 1 hour at 4°C. The precleared lysate was added to a fresh tube with 1 μg of nonspecific IgG or a specific antibody (E2F1; C-20, Santa Cruz Biotechnology; RRID: AB_631394) for 1 hour with rotation. Protein A/G beads were then added for a further hour. The beads were washed four times in RIP lysis buffer and resuspended in 400 μl of RIP lysis buffer. This was separated into two fractions—one for protein isolation and the other for RNA extraction. For protein isolation, beads were dried and resuspended in SDS-loading buffer before boiling. For RNA extraction, an equal amount of RIP extraction buffer [350 mM NaCl, 10 mM tris-HCl (pH 7.5), 10 mM EDTA, 0.1% (w/v) SDS, and 7 M urea] was added to the fraction, along with 15 μg of proteinase K, and incubated at 37°C for 30 min before RNA purification using TRIzol. RNA was DNase-treated before first-strand cDNA synthesis using random hexamers and M-MLV reverse transcriptase.

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