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Nat Protoc. 2015 Mar;10(3):475-83. doi: 10.1038/nprot.2014.114. Epub 2015 Feb 18.

MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing.

Author information

1
Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA.
2
Australian Research Council Center of Excellence in Plant Energy Biology, The University of Western Australia, Perth, Australia.
3
Department of Genetics, University of Georgia, Athens, Georgia, USA.
4
1] Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA. [2] Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, California, USA.

Abstract

Current high-throughput DNA sequencing technologies enable acquisition of billions of data points through which myriad biological processes can be interrogated, including genetic variation, chromatin structure, gene expression patterns, small RNAs and protein-DNA interactions. Here we describe the MethylC-sequencing (MethylC-seq) library preparation method, a 2-d protocol that enables the genome-wide identification of cytosine DNA methylation states at single-base resolution. The technique involves fragmentation of genomic DNA followed by adapter ligation, bisulfite conversion and limited amplification using adapter-specific PCR primers in preparation for sequencing. To date, this protocol has been successfully applied to genomic DNA isolated from primary cell culture, sorted cells and fresh tissue from over a thousand plant and animal samples.

PMID:
25692984
PMCID:
PMC4465251
DOI:
10.1038/nprot.2014.114
[Indexed for MEDLINE]
Free PMC Article

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