Enhanced reduced representation bisulfite sequencing for assessment of DNA methylation at base pair resolution.
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.
Department of Medicine, Weill Cornell Medical College; frg9015@med.cornell.edu.
Francine E
FE
Garrett-Bakelman
Department of Medicine, Weill Cornell Medical College.
Caroline K
CK
Sheridan
Department of Medicine, Weill Cornell Medical College.
Thadeous J
TJ
Kacmarczyk
Department of Medicine, Weill Cornell Medical College.
Jennifer
J
Ishii
Department of Medicine, Weill Cornell Medical College; Institute for Computational Biomedicine, Weill Cornell Medical College.
Doron
D
Betel
Department of Medicine, Weill Cornell Medical College.
Alicia
A
Alonso
Department of Physiology and Biophysics, Weill Cornell Medical College.
Christopher E
CE
Mason
Department of Pathology, University of Michigan.
Maria E
ME
Figueroa
Department of Medicine, Weill Cornell Medical College.
Ari M
AM
Melnick
D020029:Base Pairing; D001483:Base Sequence; D018899:CpG Islands; D003596:Cytosine / Q000032:analysis / Q000737:chemistry; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000737:chemistry
D013447:Sulfites; D003596:Cytosine; D004262:DNA Restriction Enzymes