H3k79me3

ChIP was performed as described previously43 (link). Briefly, fixation of cells was done with formaldehyde and DNA was subsequently sheared by sonification with a Bioruptor (high, 15 cycles: 30′′on, 30′′off) (Diagenode, Liège, Belgium). For immunoprecipitation, Dynabeads (Invitrogen) were incubated 15 min with 5 μg of specific antibodies H3K4me3 (07–473; Merck-Millipore, The Netherlands), H3K79me2 (ab3594; Abcam), H3K79me3 (ab2621; Abcam), normal rabbit IgG (ab46540; Abcam), normal mouse IgG (12–371; Merck-Millipore), anti-FLAG (f1804; Sigma-Aldrich) and anti-HA (101P-200; Covance, The Netherlands)) in 0.02% PBS–Tween-20, then unbound antibodies were washed off, and diluted sheared chromatin was added to the complex of magnetic Dynabeads-antibody (rotating overnight at 4 °C). After washing and elution with 2% SDS and 50 mmol l⁻¹ NaHCO₃, samples were treated with RNase and Proteinase K (Roche). DNA was purified using the Qiaquick DNA spin columns (Qiagen, Venlo, Netherlands) according to the protocol. Subsequently, RT–PCR was performed with AbsoluteQPCR SYBRGreenROXMix (Thermo Scientific) using specific primers (Supplementary Table 5). The % input was expressed as AE (Ct input−Ct ChIP) × Fd × 100%, where Fd is a dilution compensatory factor and AE represents the primer efficiency.

Cells were incubated with FITC- or PE-labeled CD45−, CD34−, CD133−, CD38−, CD14−, CD19−, CD3-specific and matching isotype antibodies (Miltenyi Biotec). For chromatin flow cytometry25 (link), cells were added dropwise into tubes containing 1 ml ice-cold 88% MeOH/PBS for 30 minutes. Primary antibodies (H3K4me3 (Abcam), H3K27me3 (Diagenode), H3K79me3 (Abcam) and H3K9me3 (Upstate)) were added and samples were incubated (1 h, 4 °C). For normalization, cells were stained with an H3-specific antibody (Abcam). Cells were analyzed using a FACS Canto machine (BD) running FACS Diva software (BD).

Harvested cells were incubated in RIPA buffer (50 mM Tris–HCl [pH 8.0], 150 mM NaCl, 0.1% SDS, 0.5% SDC, 1% NP-40, 1× PIC and 1 mM EDTA [pH 8.0]) with gentle agitation at 4°C for 1 h. Cell lysates were clarified by centrifugation at 18 000 g for 15 min at 4°C. After SDS-PAGE, western blotting was performed using the following antibodies: SET/TAF-Iβ (sc-133138, 1:1000), MIB1 (sc-393551, 1:1000), p21 (sc-397, 1:1000), GFP (sc-9996, 1:1000), CBX8 (sc-374332, 1:1000) and β-actin (sc-47778, 1:1000) from Santa Cruz Biotechnology (Dallas, TX, USA); H2AK119ub (8240S, 1:5000), H3K4me3 (9751S, 1:5000) and H3K27me3 (9733S, 1:5000) from Cell Signaling Technology (Danvers, MA, USA); FLAG (F3165, 1:10 000) from Sigma-Aldrich; H3K36me1 (07-548, 1:5000), H3K36me3 (07-549, 1:5000), H3K4me2 (07-030, 1:5000), H3K9me2 (07-441, 1:5000), H3K9me3 (17–625, 1:5000), H2BK120ub (17-650, 1:5000), HA (05-904, 1:5000) and H3 (05-499, 1:5000) from Merck (Rahway, NJ, USA); RAD51 (GTX70230, 1:2500) from GeneTex (Irvine, CA, USA); H3K79me1 (ab2886, 1:5000), H3K79me2 (ab3594, 1:5000), H3K79me3 (ab2621, 1:5000) and H3K36me2 (ab9049, 1:5000) from Abcam (Cambridge, UK). β-actin (ACTIN) and histone H3 were used as loading controls.