Reduced Representation Bisulfite Sequencing (RRBS)

Abstract

Reduced representation bisulfite sequencing (RRBS) enriches CpG-rich genomic regions using the MspI restriction enzyme—which cuts DNA at all CCGG sites, regardless of their DNA methylation status at the CG site—and enables the measurement of DNA methylation levels at 5% ~ 10% of all CpG sites in the mammalian genome. RRBS has been utilized in a large number of studies as a cost-effective method to investigate DNA methylation patterns, mainly at gene promoters and CpG islands. Here, we describe protocols for gel-free preparation of RRBS libraries, quality control, sequencing, and data analysis. Our protocols typically require nine cycles of polymerase chain reaction (PCR) amplification to obtain a sufficient amount of library for sequencing when 100 ng of genomic DNA is used as a starting material; moreover, it takes 3 days to complete library preparation and quality control procedures for up to eight samples.