Computational protocol: Epigenetic repression of the Igk locus by STAT5-mediated Ezh2 recruitment

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Protocol publication

[…] Rag2-/- pro-B cells were cultured for 48 h in 10 ng/ml IL-7. Chromatin from 4 × 107 cells was used for each ChIP experiment. Antibodies to STAT5 (sc-835x; Santa Cruz Biotechnology) and H3K27me3 (07-449; Millipore) were used, which yielded approximately 140 ng and 320 ng DNA, respectively. DNA libraries were prepared from the sheared chromatin (200-600bp) and were sequenced using an Illumina Genome Analyzer II. Raw sequence data were produced using the Illumina CASAVA1.7 analysis pipeline, using the “eland-extended” option. Fastq-formatted, single-end, 36 bp reads were aligned to the mm9 reference genome (National Center for Biotechnology Information build mm9_NCBI_build_37.1) using Bowtiev0.12.7 alignment software (http://bowtie-bio.sourceforge.net/index.shtml), and only reads with unique matches were retained (). MACSv1.3.7 (http://liulab.dfci.harvard.edu/MACS/) was used to identify peaks (tag size = 36, band width = 100, model fold = 2, P-value cutoff = 1 × 10-5), and HOMER software (http://biowhat.ucsd.edu/homer/chipseq/peakMotifs.html) was used for de novo prediction of motifs within the peaks. The STAT5 and H3K27me3 peaks were presented as “smoothed tag density”, where tag=sequence read, which were calculated by a program (http://compbio.med.harvard.edu/Supplements/ChIP-seq/). To identify the overlapping peaks of STAT5 and H3K27me3, sequence reads were aligned with the UCSC genome browser (http://genome.ucsc.edu). […]

Pipeline specifications

Software tools BaseSpace, ELAND, Bowtie
Application ChIP-seq analysis