TitleEnhanced reduced representation bisulfite sequencing for assessment of DNA methylation at base pair resolution.
Publication TypeJournal Article
Year of Publication2015
AuthorsGarrett-Bakelman, Francine E., Sheridan Caroline K., Kacmarczyk Thadeous J., Ishii Jennifer, Betel Doron, Alonso Alicia, Mason Christopher E., Figueroa Maria E., and Melnick Ari M.
JournalJ Vis Exp
Issue96
Paginatione52246
Date Published2015 Feb 24
ISSN1940-087X
KeywordsBase Pairing, Base Sequence, CpG Islands, Cytosine, DNA Methylation, DNA Restriction Enzymes, Humans, Molecular Sequence Data, Sequence Analysis, DNA, Sulfites
Abstract

<p>DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.</p>

DOI10.3791/52246
Alternate JournalJ Vis Exp
PubMed ID25742437
PubMed Central IDPMC4354670
Grant ListK08 CA169055 / CA / NCI NIH HHS / United States
K08CA169055 / CA / NCI NIH HHS / United States
R01NS076465 / NS / NINDS NIH HHS / United States
R01HG006798 / HG / NHGRI NIH HHS / United States
R01 HG006798 / HG / NHGRI NIH HHS / United States
R01 NS076465 / NS / NINDS NIH HHS / United States