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Control of Developmental Regulators by Polycomb in Human Embryonic Stem Cells
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RNA polymerase II-bound genomic DNA was isolated from whole cell lysate using 8WG16, a mouse monoclonal antibody (Thompson et al., 1989). This antibody preferentially binds a form of RNA polymerase II that lacks phosphorylation at the C-terminal domain of the largest subunit of polymerase (Cho et al., 2001; Jones et al., 2004; Patturajan et al., 1999). Suz12-bound genomic DNA was isolated from whole cell lysate with a Suz12 rabbit polyclonal antibody purchased from Abcam (AB12201) (Kirmizis et al., 2004) or Upstate (07-379). When possible, immunprecipitations were performed with both antibodies to increase confidence in the targets identified. Eed-bound genomic DNA was isolated from whole cell lysate using an Eed mouse monoclonal antibody previously described (Hamer et al., 2002). H3-K27me3-bound genomic DNA was isolated from whole cell lysate using rabbit polyclonal antibody purchased from Abcam (AB6002). Chromatin immunoprecipitations against H3K27me3 were compared to reference DNA obtained by chromatin immunoprecipitation of total histone H3 (Abcam AB1791; epitope derived from Cterminal 100 amino acids of histone H3) to normalize for nucleosome density. |
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