Core Transcriptional Regulatory Circuitry in Human Embryonic Stem Cells

Mapping Transcription Factor Occupancy in Human Embryonic Stem Cells

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

• Download Data
• Data Analysis

The Nanog (AF1997 R&D Systems) and Sox2 (AF2018 R&D Systems) antibodies used in this study were immunoaffinity purified against the human protein and shown to recognize their target protein in Western blots and by immunocytochemistry (R&D Systems Minneapolis, MN). Multiple Oct4 antibodies directed against different portions of the protein were used for location analysis (AF1759 R&D Systems, sc-8628 Santa Cruz, sc-9081 Santa Cruz), some of which were immunoaffinity purified and have been shown to recognize their target protein in Western blots and by immunocytochemistry. Prior to conducting the experiments with Agilent arrays, we compared these three Oct4 antibodies by performing location analysis with self-printed promoter arrays and found that they performed similarly. ChIP experiments carried out with AF1759 and sc-8628 were hybridized to the Agilent 10-array sets. In addition, our immunofluorescence results indicated that a nuclear protein was detected only in undifferentiated ES cells with the Oct4 antibody (sc-9081) (Figure S3; compare ES cell with MEF). E2F4 antibodies (sc-1082) were obtained from Santa Cruz Biotech and have been shown to specifically recognize previously reported E2F4 target genes (Table S2) (Ren et al., 2002; Weinmann et al., 2002). The Oct4 antibodies used in this analysis show Oct4-specific staining in ES cell nuclei (Figure S3).