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Development and application of ChIPSeq: genome‐wide DNA binding by MyoD
Author(s) -
Kwan Gordon,
Sharp Sandra B,
Wold Barbara J
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.797.2
Subject(s) - myod , biology , chromatin immunoprecipitation , dna binding site , chip sequencing , genetics , myogenesis , computational biology , chip on chip , transcription factor , microbiology and biotechnology , gene , promoter , gene expression , chromatin remodeling
ChIPSeq is an application of high throughput direct sequencing (Solexa / Illumina) to assay for factor‐DNA binding sites retrieved by chromatin immunoprecipitation (ChIP). A goal was to achieve reliable identification of true binding sites throughout large genomes, without need for cloning or microarray. The target protein MyoD, a muscle‐specific transcription factor, was chosen for proof‐of‐principle for the large class of factors that bind small and degenerate motifs (e.g. CANNTG) and to acquire new information regarding the role of MyoD in myogenesis. For ChIP optimization, MyoD‐ChIP DNA fragments were assayed by quantitative (real time) PCR using a set of locations known to be positive for MyoD binding. For the direct sequencing assay, MyoD‐ChIP and control normal mouse IgG‐ChIP fragments were cluster‐amplified and sequenced. 32‐nt sequence reads were mapped to the mouse genome. MyoD‐ChIP and control reads were analyzed jointly to identify enriched regions. 6.8M out of 17.4M total sequence reads mapped to single locations. 5,113 genes were identified from 13,194 enriched regions. Data revealed expected and unexpected MyoD binding site locations. The results suggest that the method is broadly applicable for identifying binding locations and thereby identifying binding sequence motifs of any transcription factor in any sequenced genome, large or small. CSUPERB Joint Venture Grant to SBS.