Project description:Commercially available histone antibodies were analyzed using peptide microarrays to assess specificity. For two H3K27me3 antibodies from different vendors, we performed ChIP-seq in WT and EED-null murine ES cells to assess specificity. Cell lines were generated from e3.5 blastocysts of EED fl/fl animals and transfected with plasmid CAG-CRE-ERT2. Null cells were generated by single cell clones from tamoxifen-treated parent cell line.
Project description:Commercially available histone antibodies were analyzed using peptide microarrays to assess specificity. For two H3K27me3 antibodies from different vendors, we performed ChIP-seq in WT and EED-null murine ES cells to assess specificity. Cell lines were generated from e3.5 blastocysts of EED fl/fl animals and transfected with plasmid CAG-CRE-ERT2. Null cells were generated by single cell clones from tamoxifen-treated parent cell line. Analysis of the specificity of two H3K27me3 antibodies using WT and EED (Embryonic Ectoderm Develepment)-null (mutants lacking H3K27me3) ES cells. The goal was to compare WT and KO for each antibody with the expectation that if the antibodies were specific, there would remain few peaks and little signal in the knockout. We sequenced 2 replicates of each condition (3 for WT input) and after comparing replicates, we merged data, called peaks, and calculated signal in bins centered on the wild type H3K27me3 peaks. As little to no signal remained either by direct comparison of peaks or looking at 'metagenes' of the peaks, we concluded that the antibodies were specific. Antibodies: Abcam H3K27me3 (ab6002, lot# GR130002-1) Upstate H3K27me3 (07-449, lot# 2455635)
