H2ak119ub

Histone modification and PolII ChIP was performed as previously described^{17 (link),62 (link)}, respectively. Immunoprecipitation was done with antibodies: anti-H3 (Abcam, #ab1791), anti-H3K27me3 (Millipore, #07-449), H2AK119ub (Cell Signaling Technology, #8240), anti-H3K36me3 (Abcam, #ab9050). PolII antibodies were as in^{64 (link)}. All ChIP experiments were followed by qPCR with indicated primer pairs (Supplementary Table 2).

0.25–5 μg of acid extracted histone lysate was prepared with Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, CA, USA) and β-mercaptoethanol (Bio-Rad Laboratories) and heated at 95°C for 5 min before transferring to ice to cool. Samples were then separated by SDS-PAGE on an 8–16% Mini-PROTEAN TGC Precast Protein Gel (Bio-Rad Laboratories) at 100 V for 60 min. Samples were transferred to 0.2 μM nitrocellulose membranes (Bio-Rad Laboratories) at 65 V for 45 min at 4°C and blocked in 5% milk in TBST (TBS with 0.1% Tween-20 (MilliporeSigma)). Blots were then incubated overnight in primary antibody diluted in milk with rotation at 4°C. The following antibodies were used for these studies: H2AK119Ub (8240S; Cell Signaling), H3K27me3 (07-449; Millipore), H3 (07-690; Upstate), H4 (ab7311; abcam), Flag (A8592; Sigma), H3K9me2 (ab1220; abcam), and H3K9me3 (8898; abcam). Blots were incubated in anti-mouse IgG (NA931; Millipore Sigma) or anti-rabbit (NA934; Millipore Sigma) for 1 h at room temperature. Immobilon Western Chemiluminescent HRP Substrate (MilliporeSigma) was used to visualize bands with a Konica SRX-101 X-ray film processor.

ChIP assays were carried out on ∼2–5 million cells per sample and per epitope, following the procedures described previously (Mikkelsen et al, 2007 (link)). In brief, chromatin from formaldehyde-fixed cells was fragmented to a size range of 200–700 bases with a Branson 250 sonifier. Solubilized chromatin was immunoprecipitated with the indicated antibodies overnight at 4°C. Antibody-chromatin complexes were pulled down with protein G-Dynabeads (Life Technologies), washed, and then eluted. After cross-link reversal, RNase A, and proteinase K treatment, immunoprecipitated DNA was extracted with AMP Pure beads (Beckman Coulter). ChIP DNA was quantified with Qubit. 1–5 ng ChIP DNA samples were used to prepare sequencing libraries, and ChIP DNA and input controls were sequenced with the Nextseq 500 Illumina genome analyzer.
Antibodies used for these studies were SMARCA2/4 (39805; Active Motif), H3K4me1 (ab8895; Abcam), H3K4me3 (07-473; Millipore), H3K9ac (ab4441; Abcam), H3K27ac (39133; Active Motif), H3K27me3 (07-449; Millipore), H3K36me3 (ab9050; Abcam), V5 (ab15828; Abcam), RING1B (5694; Cell Signaling), H2AK119ub (8240; Cell Signaling), RYBP (59451204; Sigma-Aldrich), and USP7 (A300-033A; Bethyl Laboratories).

Antibodies against EZH1/2, SUZ12, JARID2, and EED were previously characterized^{33 (link),63 (link)}. RBAP48 mouse mAb (GWB-C12FDE) was purchased from GenWay Biotech; H3 mAb (39163) and H3K27me2 mAb (61435) were purchased from Active Motif. Polyclonal rabbit one against H3 from Cell Signaling Technology (9715); H3K9me2 (ab1220) and H3K27Ac (ab 4729) were purchased from Abcam; H3K27me1 mouse mAb C0321 from Active Motif; H3K27me3 Rabbit mAb C36B11 (9733), H3K4me3 Rabbit mAb C42D8 (9751), Rabbit mAb D7C6X (14129), Rabbit mAb H2AK119ub (8240 S) and mouse mAb10E2 HDAC1 (5356 S) from Ozyme (Cell Signaling Technology). hEZHIP (HPA006128) and mAb Flag-M2 (F1804) were purchased from SIGMA; Anti-Germ cell-specific Rabbit Polyclonal DPP3A/Stella (19878) and TRA98 (Ab82527) Rat monoclonal one from Abcam. For Immunofluorescence, EED was detected with the M26 antibody. Antibody against mEZHIP was raised against the two following synthetic peptides: CAESSRAESDQSSPAG (corresponding to a.a. 91–106) and CAQSAGRNLRPRPRSS (corresponding to a.a. 192–206). Anti-mouse β-TUBULIN was purchased from Invitrogen 32–2600.
Primary antibodies were diluted 1:3000 for WB analysis and 1:250 for Immunostaining.