Immunofluorescence Staining of ESCs

ESCs were grown on slides without feeders, fixed with 2% formaldehyde for 15 min, permeabilized with 0.4% Triton X-100 for 5 min and blocked with 0.2% fish gelatin (Sigma) for 30 min. Slides were incubated with primary antibody for 2 h (diluted in 0.2% fish gelatin and 5% normal goat serum), washed three times and incubated with Alexa-fluor conjugated secondary antibody for 2 h (Life Technologies). After washing five times, the slides were stained with DAPI (1 μg ml⁻¹), and mounted using mounting media (Dako). Primary antibodies used were protein-A purified anti-AEBP2 (ref. 36 (link)) at 1:10 dilution, anti-H3K27me3 (Active motif 39157) at 1:500 dilution, anti-SUZ12 (Cell Signalling 3737) at 1:500 dilution, anti-EZH2 (Cell Signalling 5246) at 1:500 dilution, anti-EED (a kind gift from A. Otte) at 1:100 dilution, anti-JARID2 (Novus Biologicals NB100-2214) at 1:500 dilution, H2AK119u1 (NEB 8240) at 1:500 dilution, anti-FLAG (Sigma M2 F1804 and F7425) at 1:500 dilution, anti-mCherry (Source Bioscience ABE3523 and gift from F. Barr) at 1:500 and 1:800 dilution, anti-RING1B at 1:500 dilution and anti-ATRX (a gift from R. Gibbons) at 1:10 dilution.