Cells for ChIP-seq were cross-linked with 1% formaldehyde for 10 min at RT and quenched with 125 mM glycine for 5 min. Cells were collected and washed once with cold TBS. EBs were digested by 0.25% trypsin (Gibco, Gaithersburg, Maryland) at 37°C before fixation using 1% formaldehyde for 10 min at RT. Fixed cells were re-suspended in lysis buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 0.5% IGEPAL-CA630) and incubated on ice for 10 min. After centrifugation, the cell pellet was washed once with MNase digestion buffer (20 mM Tris-HCl, pH7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2). After re-suspended in 250 µl MNase digestion buffer with proteinase inhibitors, 2000 units per 4x106 cells MNase (M0247S; New England Biolabs, Ipswitch, Massachusetts) was added. After incubation at 37°C for 20 min, 250 µl of STOP buffer (100 mM Tris-HCl, pH8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% sodium deoxycholate) was added. Lysates were sonicated for 15 cycles (30 sec on and 30 sec off) in Bioruptor (Diagenode, Inc., Danville, New Jersey) and centrifuged at 21,130 x g for 10min. the supernatant was collected after centrifugation and chromatin content was estimated by the Qubit assay (Invitrogen). The chromatin was incubated with 5 µg anti-H3K27me3 antibody (Cell Signaling, Cat.# 9733), 2 µg anti-H3K4me3 antibody (Abcam, ab8580) at 4°C overnight on a rotator. 30 µl Protein-G Mag Sepharose beads (GE Healthcare, Little Chalfont, United Kingdom) was added and incubated for additional 3 hours at 4°C. Beads were washed with ChIP buffer (50 mM Tris-HCl, pH8.0, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), high salt buffer (ChIP buffer with 500 mM NaCl), LiCl buffer (10 mM Tris-HCl, pH8.0, 0.25 M LiCl2, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), and TE buffer. Chromatin on beads was eluted and reverse-crosslinked at 65°C overnight and was purified by PCR purification kit (Qiagen) after the treatment of RNase A and proteinase K. ChIP-seq libraries were prepared from 10 ng ChIP and input DNA using the Ovation ultralow DR Multiplex kit (NuGEN). The ChIP-seq libraries were sequenced at single end on an Illumina HiSeq 2000 instrument.