title: Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling.
abstract: Genome-wide mapping of 5-methylcytosine is of broad interest to many fields of biology and medicine. A variety of methods have been developed, and several have recently been advanced to genome-wide scale using arrays and next-generation sequencing approaches. We have previously reported reduced representation bisulfite sequencing (RRBS), a bisulfite-based protocol that enriches CG-rich parts of the genome, thereby reducing the amount of sequencing required while capturing the majority of promoters and other relevant genomic regions. The approach provides single-nucleotide resolution, is highly sensitive and provides quantitative DNA methylation measurements. This protocol should enable any standard molecular biology laboratory to generate RRBS libraries of high quality. Briefly, purified genomic DNA is digested by the methylation-insensitive restriction enzyme MspI to generate short fragments that contain CpG dinucleotides at the ends. After end-repair, A-tailing and ligation to methylated Illumina adapters, the CpG-rich DNA fragments (40-220 bp) are size selected, subjected to bisulfite conversion, PCR amplified and end sequenced on an Illumina Genome Analyzer. Note that alignment and analysis of RRBS sequencing reads are not covered in this protocol. The extremely low input requirements (10-300 ng), the applicability of the protocol to formalin-fixed and paraffin-embedded samples, and the technique's single-nucleotide resolution extends RRBS to a wide range of biological and clinical samples and research applications. The entire process of RRBS library construction takes ∼9 d.
authors.affiliation: Broad Institute, Cambridge, Massachusetts, USA.
authors.forename: Hongcang
authors.initials: H
authors.lastname: Gu
authors.forename: Zachary D
authors.initials: ZD
authors.lastname: Smith
authors.forename: Christoph
authors.initials: C
authors.lastname: Bock
authors.forename: Patrick
authors.initials: P
authors.lastname: Boyle
authors.forename: Andreas
authors.initials: A
authors.lastname: Gnirke
authors.forename: Alexander
authors.initials: A
authors.lastname: Meissner
mesh_terms: D018899:CpG Islands; D019175:DNA Methylation; D015723:Gene Library; D023281:Genomics / Q000379:methods; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000737:chemistry
chemical_list: D013447:Sulfites; C042345:hydrogen sulfite