ChIP was performed for SUZ12 (Abcam), EZH2 (Cell Signaling), JARID2 (Cell Signalling), H3K27me3 (Abcam), total H3 (Abcam), and EP300 (Bethyl) as previously described (Kanhere et al. 2012 (link)), with variations described in the Supplemental Methods . Enrichment of specific gene sequences was measured by qPCR (Applied Biosystems). Libraries were sequenced using an Illumina HiSeq 2500 (50 bp single-end). ChIP-seq reads were trimmed to remove adaptors and low-quality bases and aligned with Bowtie 2 (Langmead and Salzberg 2012 (link)). MACS14 (Zhang et al. 2008 (link)) and MAnorm (Shao et al. 2012 (link)) were used to identify sites at which SUZ12 binding was significantly changed by RNase treatment (P < 0.05).
Protocol detail
ChIP-seq analysis of chromatin modifications
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Corresponding Organization :
Other organizations : University College London, National Hospital for Neurology and Neurosurgery, Imperial College London
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Variable analysis
independent variables
- RNase treatment
- SUZ12 binding sites
- ChIP performed for SUZ12 (Abcam), EZH2 (Cell Signaling), JARID2 (Cell Signalling), H3K27me3 (Abcam), total H3 (Abcam), and EP300 (Bethyl) as previously described (Kanhere et al. 2012)
- Variations described in the Supplemental Methods
- None specified
- None specified
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