Control of Developmental Regulators by Polycomb in Human Embryonic Stem Cells

Mapping RNA Polymerase II Occupancy in Embryonic Stem Cells

• Human ES Cells
• Growth Conditions
• Immunohistochemical Analysis of Pluripotency Markers
• Teratoma Formation
• Embryoid Bodies (EB)
• Technology and Protocols
• Antibodies
• Chromatin Immunoprecipitations
• Microarray Design

• Download Data
• Data Analysis

ES cells were harvested by enzymatic digestion and EBs were allowed to form by plating ~1 X 10^6 cells/well in suspension on 6-well non-adherent, low cluster dishes for 30 days. EBs were grown in the absence of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (FGF) in culture medium containing 2x serum replacement. EBs were then harvested, fixed for 30 minutes in 4% paraformaldehyde at 4°C, and placed in 30% sucrose overnight prior to embedding the tissue in O.C.T. freezing compound (Tissue-Tek). Cryosections were obtained as described for teratoma formation. Confocal images were obtained for all three germ layer markers again confirming that the H9 cells used in our analysis have maintained differentiation potential (data not shown; results similar to those shown in Figure S3).