This track displays maps of chromatin state generated by the Broad/MGH ENCODE group using ChIP-seq. Chemical modifications (methylation, acetylation) to the histone proteins present in chromatin influence gene expression by changing how accessible the chromatin is to transcription.
The ChIP-seq method involves first using formaldehyde to cross-link histones and other DNA-associated proteins to genomic DNA within cells. The cross-linked chromatin is subsequently extracted, mechanically sheared, and immunoprecipitated using specific antibodies. After reversal of cross-links, the immunoprecipitated DNA is sequenced and mapped to the human reference genome. The relative enrichment of each antibody-target (epitope) across the genome is inferred from the density of mapped fragments.
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Peaks and signals displayed in this track are the results of pooled replicates. The raw sequence and alignment files for each replicate are available for download.
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ChIP-seq: Cells were grown according to the approved ENCODE cell culture protocols. Cells were fixed in 1% formaldehyde and resuspended in lysis buffer. Chromatin was sheared to 200-700 bp using a Diagenode Bioruptor. Solubilized chromatin was immunoprecipitated with antibodies against each of the histone antibodies listed above. Antibody-chromatin complexes were pulled down using protein A-sepharose (or anti-IgM-conjugated agarose for RNA polymerase II), washed and then eluted. After cross-link reversal and proteinase K treatment, immunoprecipitated DNA was extracted with phenol-chloroform, ethanol precipitated, treated with RNAse and purified. A quantity of 1-10 ng of DNA was end-repaired, adapter-ligated and sequenced by Illumina Genome Analyzers as recommended by the manufacturer.
Alignment: Sequence reads from each IP experiment were aligned to the
human reference genome (GRCh37/hg19) using
MAQ
with default parameters, except '-C 11
' and
'-H output_file
' were added. These options output up to 11 additional best matches
for each read (if any are found) to a file. This information was used to filter
out any read that had more than 10 best matches on the genome. Note that it is
likely that instances where multiple reads align to the same position and with
the same orientation are due to enhanced PCR amplification of a single DNA
fragment. No attempt has been made, however, to remove such artifacts from the
data, following ENCODE practices.
Signal: Fragment densities were computed by counting the number of
reads overlapping each 25 bp bin along the genome. Densities were computed using
igvtools count
with default parameters (in particular, '-w 25
' to set window size
of 25 bp and '-f mean
' to report the mean value across the window),
except for '-e
' which was set to extend the reads to 200 bp, and the
.wig output was converted to bigWig using wigToBigWig
from the UCSC Kent software package.
Peaks: Discrete intervals of ChIP-seq fragment enrichment were
identified using Scripture,
a scan statistics approach, under the assumption of uniform background signal.
All data sets were processed with '-task chip
', and with
'-windows 100,200,500,1000,5000,10000,100000
' (no mask file nor
the '-trim
' option have been used). The resulting called segments
were then further filtered to remove intervals that were significantly enriched
only because they contain smaller enriched intervals within them. This
post-processing step has been implemented using Matlab
. The use of the
post-processing step allowed very large enriched intervals (of the order of
Mbps for H3K27me3, for instance) to be detected, as well as much smaller
intervals, without the need to tailor the parameters of Scripture
based on prior expectations.
This is Release 3 (Aug 2012). It contains 83 new experiments including 6 new cell lines and 25 new antibodies. Please note that an antibody previously labeled "Pol2 (b)" is, in fact, Covance antibody MMS-128P with the target POLR2A.
The ChIP-seq data were generated at the Broad Institute and in the Bernstein lab at the Massachusetts General Hospital/Harvard Medical School.
Data generation and analysis were supported by funds from the NHGRI, the Burroughs Wellcome Fund, Massachusetts General Hospital and the Broad Institute.
Contact: Noam ShoreshBernstein BE, Kamal M, Lindblad-Toh K, Bekiranov S, Bailey DK, Huebert DJ, McMahon S, Karlsson EK, Kulbokas EJ 3rd, Gingeras TR et al. Genomic maps and comparative analysis of histone modifications in human and mouse. Cell. 2005 Jan 28;120(2):169-81.
Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, Fry B, Meissner A, Wernig M, Plath K et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell. 2006 Apr 21;125(2):315-26.
Ernst J, Kheradpour P, Mikkelsen TS, Shoresh N, Ward LD, Epstein CB, Zhang X, Wang L, Issner R, Coyne M et al. Mapping and analysis of chromatin state dynamics in nine human cell types. Nature. 2011 May 5;473(7345):43-9.
Guttman M, Garber M, Levin JZ, Donaghey J, Robinson J, Adiconis X, Fan L, Koziol MJ, Gnirke A, Nusbaum C et al. Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nat Biotechnol. 2010 May;28(5):503-10.
Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, Giannoukos G, Alvarez P, Brockman W, Kim TK, Koche RP et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature. 2007 Aug 2;448(7153):553-60.
Ram O, Goren A, Amit I, Shoresh N, Yosef N, Ernst J, Kellis M, Gymrek M, Issner R, Coyne M et al. Combinatorial patterning of chromatin regulators uncovered by genome-wide location analysis in human cells. Cell. 2011 Dec 23;147(7):1628-39.
Data users may freely use ENCODE data, but may not, without prior consent, submit publications that use an unpublished ENCODE dataset until nine months following the release of the dataset. This date is listed in the Restricted Until, above. The full data release policy for ENCODE is available here.