title: MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing.
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.
authors.affiliation: Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Urich
authors.affiliation: Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Nery
authors.affiliation: Australian Research Council Center of Excellence in Plant Energy Biology, The University of Western Australia, Perth, Australia.
authors.forename: Ryan
authors.initials: R
authors.lastname: Lister
authors.affiliation: Department of Genetics, University of Georgia, Athens, Georgia, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Schmitz
authors.affiliation: 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.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Ecker
mesh_terms: D003596:Cytosine / Q000737:chemistry*; D053938:DNA Fragmentation; D019175:DNA Methylation / Q000235:genetics*; D015698:Genomic Library; D023281:Genomics / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000737:chemistry
chemical_list: D013447:Sulfites; D003596:Cytosine; C042345:hydrogen sulfite