ChIP-Seq Analysis of Transcription Factors

ChIP was performed as previously described (22 (link),36 (link),37 (link)) using the following antibodies: AR N20 [SC-816X, Santa Cruz], GABPα [SC-22810, Santa Cruz], ERG [SC-353, Santa Cruz]. Biological replicates were used. Enrichment was tested with 6 μl DNA by real-time PCR using SYBRgreen (Applied Biosystems). Single-end SOLEXA libraries were prepared as previously described (36 (link)) and 36 bp sequence reads were generated by the Illumina HiSeq 2000. Sequence reads were aligned against the Human Reference Genome (assemby hg18, NCBI Build 36 (link)) using Burrows-Wheeler Aligner (BWA) version 0.5.5 (38 (link)). Reads were filtered by removing those with a BWA alignment quality score less than 15 as well as duplicate reads. Enriched regions of the genome were identified by comparing ChIP samples to input samples using MACS (39 (link)) and SWEMBL (http://www.ebi.ac.uk/∼swilder/SWEMBL/). Only peaks that were identified by both MACS and SWEMBL (high confidence peaks) were used for further analyses.

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Sharma N.L., Massie C.E., Butter F., Mann M., Bon H., Ramos-Montoya A., Menon S., Stark R., Lamb A.D., Scott H.E., Warren A.Y., Neal D.E, & Mills I.G. (2014). The ETS family member GABPα modulates androgen receptor signalling and mediates an aggressive phenotype in prostate cancer. Nucleic Acids Research, 42(10), 6256-6269.

Publication 2014

GABPα SYBRgreen HiSeq 2000

Antibodies Biological Chip Genome Genome human Real time pcr

Corresponding Organization : University of Cambridge

Other organizations : Max Planck Institute of Biochemistry, University of Oslo, Oslo University Hospital

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Variable analysis

independent variables

Antibodies used for ChIP: AR N20 [SC-816X, Santa Cruz], GABPα [SC-22810, Santa Cruz], ERG [SC-353, Santa Cruz]

dependent variables

Enrichment tested by real-time PCR using SYBRgreen
Sequence reads generated by Illumina HiSeq 2000
Enriched regions of the genome identified by comparing ChIP samples to input samples using MACS and SWEMBL

control variables

Biological replicates were used
Sequence reads were aligned against the Human Reference Genome (assembly hg18, NCBI Build 36)
Reads were filtered by removing those with a BWA alignment quality score less than 15 as well as duplicate reads
Only peaks that were identified by both MACS and SWEMBL (high confidence peaks) were used for further analyses

controls

Input samples used as a control for identifying enriched regions of the genome

Annotations

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