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Nat Protoc. 2014 Sep;9(9):2180-93. doi: 10.1038/nprot.2014.146. Epub 2014 Aug 21.

A robust chromatin immunoprecipitation protocol for studying transcription factor-DNA interactions and histone modifications in wood-forming tissue.

Author information

1
1] State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China. [2] Forest Biotechnology Group, Department of Forestry &Environmental Resources, North Carolina State University, Raleigh, North Carolina, USA.
2
1] State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China. [2] Forest Biotechnology Group, Department of Forestry &Environmental Resources, North Carolina State University, Raleigh, North Carolina, USA. [3] College of Forestry, Shandong Agricultural University, Taian, Shandong, China.
3
Forest Biotechnology Group, Department of Forestry &Environmental Resources, North Carolina State University, Raleigh, North Carolina, USA.
4
State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China.
5
1] State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, China. [2] Forest Biotechnology Group, Department of Forestry &Environmental Resources, North Carolina State University, Raleigh, North Carolina, USA. [3] Department of Forest Biomaterials, North Carolina State University, Raleigh, North Carolina, USA.

Abstract

Woody cells and tissues are recalcitrant to standard chromatin immunoprecipitation (ChIP) procedures. However, we recently successfully implemented ChIP in wood-forming tissue of the model woody plant Populus trichocarpa. Here we provide the detailed ChIP protocol optimized for wood-forming tissue that we used in those studies. By using stem-differentiating xylem (SDX; a wood-forming tissue), we identified all steps that were ineffective in standard ChIP protocols and systematically modified them to develop and optimize a robust ChIP protocol. The protocol includes tissue collection, cross-linking, nuclear isolation, chromatin extraction, DNA fragmentation, immunoprecipitation, DNA purification and sequence analysis. The protocol takes 2.5 d to complete and allows a robust 8-10-fold enrichment of transcription factor (TF)-bound genomic fragments (~150 ng/g of SDX) over nonspecific DNAs. The enriched DNAs are of high quality and can be used for subsequent PCR and DNA-seq analyses. We used this protocol to identify genome-wide specific TF-DNA interactions during wood formation and histone modifications associated with regulation of wood formation. Our protocol, which may be suitable for many tissue types, is so far the only working ChIP system for wood-forming tissue.

PMID:
25144269
DOI:
10.1038/nprot.2014.146
[Indexed for MEDLINE]

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