Protein coated a g agarose beads

For ChIP analysis, cells were fixed with 1% formaldehyde for 15 min at room temperature, and then quenched with 2.5 M glycine. Subsequently, the cells were lysed on ice for 10 min in SDS buffer containing protease inhibitors, and then sonicated with Sonics Vibra Cell^TM (Sonics & Materials Inc., CT, USA; 7 min: 10 s pulse, 10 s interval) on ice. The fragmented chromatin samples were centrifuged at 8000 ×g at 4 °C for 5 min and the supernatant was collected. The samples were pre-cleared, and then incubated overnight at 4 °C with the appropriate antibodies and protein-coated A/G agarose beads (Santa Cruz) with gentle shaking. The immunoprecipitated eluates were reverse cross-linked and recovered through DNA purification for PCR. Anti-H3K4me3 (ab1012; Abcam), anti-H3K9ac (ab12179; Abcam), anti-H3K27me3 (ab6002; Abcam), anti-EZH2 (#5246S; Cell Signaling), anti-SUZ12 (#3737; Cell Signaling), anti-EED (ab96801; Abcam), anti-UTX (ab36938; Abcam), anti-JMJD3 (ab169197; Abcam), and non-immune mouse IgG (sc2025; Santa Cruz) antibodies were used. ChIP-PCR primers are listed in Table 2.

Cells were washed twice with ice-cold PBS and then resuspended in Cell Lysis/IP Buffer (Thermo Scientific, MA, USA) supplemented with protease and RNase inhibitor. The lysates were centrifuged at 12,000× g for 15 min at 4 °C to remove cell debris after incubation on ice for 30 min at 4 °C. Next, an appropriate amount of the antibody was added into the supernatant and incubated overnight at 4 °C on a rotator. Subsequently, 30 µL of Protein A/G-Coated Agarose Beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added into the supernatant, which was incubated at 4 °C for 1 h, followed by three washes in Wash Buffer. Finally, 0.5 mL of Trizol was added to the tube, then RNA was extracted following the manufacturer’s instructions and subjected to RT-PCR analysis for quantification.

Immunoprecipitation assay was performed by cell lysis on ice in immunoprecipitation buffer (25 mM Tris HCl, pH 7.5, 150 mM KCl, 5 mM MgCl2, 1 mM EGTA,
10% glycerol, 0.8% Igepal/NP40, 0,4 U/μl RNAsi OUT and protease inhibitors cocktail from Roche Diagnostics, Basel, Switzerland). The lysates were cleared by centrifugation and quantified by Bradford protein assay (Bio-Rad Laboratories Hempstead, UK). Equivalent amounts (200 μg) of protein were incubated at 4°C with rotation overnight in immunoprecipitation buffer with 3 μg of anti-MDM2 (H-221; Santa Cruz Biotechnology, Santa Cruz, CA, USA) Protein A/G-coated agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added to the extracts and mixed by rotation for an additional 3 hours at 4°C. The beads were washed four times with immunoprecipitation buffer. At the end of the final wash, beads were resuspended in Laemmli loading buffer for western blot analysis or in TRIreagent (Ambion, Austin, TX, USA) for RNA extraction. The extracted RNA was divided in two fractions and used directly for total 5S rRNA analysis or to retrive the 5-EU labeled and the unlabeled RNA fraction by using the nascent RNA capture kit^® (Life technologies, Eugene Oregon, USA), as indicated after.