## Description

This super-track displays related tracks of open chromatin from the Duke/UNC/Texas/EBI ENCODE group. We identified open chromatin using two independent and complementary methods: DNaseI hypersensitivity (HS) and Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE), combined with chromatin immunoprecipitation (ChIP) for select regulatory factors. Each method is verified by two detection platforms: Illumina (formerly Solexa) sequencing by synthesis, and high-resolution 1% ENCODE tiled microarrays supplied by NimbleGen.

DNaseI HS data: DNaseI is an enzyme that has long been used to map general chromatin accessibility and the DNaseI "hyperaccessibility" or "hypersensitivity" is a feature of active cis-regulatory sequences. The use of this method has led to the discovery of functional regulatory elements that include enhancers, silencers, insulators, promotors, locus control regions and novel elements. DNaseI hypersensitivity signifies chromatin accessibility following binding of trans-acting factors in place of a canonical nucleosome.

FAIRE data: FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements) is a method to isolate and identify nucleosome-depleted regions of the genome. FAIRE was initially discovered in yeast, but subsequently was shown to also identify active regulatory elements in human cells (Giresi et al., 2006). Most DNA fragments isolated by FAIRE are 200 bp in length.

ChIP data: ChIP (Chromatin Immunoprecipitation) is a method to identify the specific location of proteins that are directly or indirectly bound to genomic DNA. By identifying the binding location of sequence-specific transcription factors, general transcription machinery components, and chromatin factors, ChIP can help in the functional annotation of the open chromatin regions identified by DNaseI HS mapping and FAIRE.

## Methods

DNaseI hypersensitive sites were isolated using methods called DNase-seq or DNase-chip (Boyle et al., 2008, Crawford et al., 2006). Briefly, cells were lysed with NP40 and intact nuclei were digested with optimal levels of DNaseI enzyme. DNaseI digested ends were captured from three different DNase concentrations and material was sequenced using Illumina (Solexa) sequencing. DNase-seq data was verified using material that was hybridized to NimbleGen Human ENCODE tiled array (1% of the genome).

FAIRE was performed (Giresi et al., 2007) by cross-linking proteins to DNA using 1% formaldehyde solution, the complex was sheared using sonication, and a phenol/chloroform extraction was performed to remove DNA fragments cross-linked to protein. The DNA recovered in the aqueous phase was sequenced using the Illumina sequencing and hybridized to NimbleGen Human ENCODE arrays (1% of the genome).

To perform ChIP, proteins were cross-linked to DNA in vivo using 1% formaldehyde solution (Bhinge et al., 2007, ENCODE Project Consortium., 2007). Cross-linked chromatin was sheared by sonication and immunoprecipitated using a specific antibody against the protein of interest. After reversal of the cross-links, the immunoprecipitated DNA was used to identify the genomic location of transcription factor binding. This was accomplished by Solexa sequencing of the ends of the immunoprecipitated DNA (ChIP-seq) as well as labeling and hybridization of the immunoprecipitated DNA to NimbleGen Human ENCODE tiling arrays (1% of the genome) along with the input DNA as reference (ChIP-chip).

### High-Throughput Sequence Data Analysis:

ENCODE Array Data Analysis: Discrete DNase HS, FAIRE, and ChIP sites were identified from DNase/FAIRE/ChIP-seq by setting a parzen cutoff based on ROC curve analysis using peaks and non-peaks identified from DNase/FAIRE/ChIP-chip using 1% ENCODE NimbleGen arrays. Tiled array peaks were identified using ChIPOTle (Buck et al., 2005), and non-peaks were identified using less stringent peak calling cutoffs.

## Credits

These data and annotations were created by a collaboration of multiple institutions:

Duke University's Institute for Genome Sciences & Policy (IGSP) Alan Boyle, Lingyun Song, Terry Furey, and Greg Crawford

University of North Carolina at Chapel Hill Paul Giresi and Jason Lieb

Universty of Texas at Austin Zheng Liu, Ryan McDaniell, Bum-Kyu Lee, and Vishy Iyer

European Bioinformatics Insitute Paul Flicek, Stefan Graf, Damian Keefe, and Ewan Birney

We thank NHGRI for ENCODE funding support.

## References

The ENCODE Project Consortium. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 2007 447: 799-816 Retrieved from "http://encodewiki.ucsc.edu/EncodeDCC/index.php/CrawfordDNaseDoc"

Bhinge, A.A., Kim, J., Euskirchen, G.M., Snyder, M., & Iyer, V.R. Mapping the chromosomal targets of STAT1 by Sequence Tag Analysis of Genomic Enrichment (STAGE). Genome Res. 2007 17: 910-916.

Boyle AP, Davis S, Shulha HP, Meltzer P, Margulies EH, Weng Z, Furey TS, Crawford GE. High-resolution mapping and characterization of open chromatin across the genome. Cell. 2008 Jan 25;132(2):311-22.

Buck MJ, Nobel AB, Lieb JD, ChIPOTle: a user-friendly tool for the analysis of ChIP-chip data. Genome Biology. 2005

Crawford GE, Davis S, Scacheri PC, Renaud G, Halawi MJ, Erdos MR, Green R, Meltzer PS, Wolfsberg TG, Collins FS. DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays. Nat Methods. 2006 Jul;3(7):503-9.

Crawford GE, Holt IE, Whittle J, Webb BD, Tai D, Davis S, Margulies EH, Chen Y, Bernat JA, Ginsburg D, Zhou D, Luo S, Vasicek TJ, Daly MJ, Wolfsberg TG, Collins FS. Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS). Genome Res. 2006 Jan;16(1):123-31.

Giresi PG, Kim J, McDaniel RM, Iyer VR, Lieb JD. FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolated active regulatory elements in human chromatin. Genome Res. 2007;17:877-885.