How does reduced representation bisulfite sequencing (RRBS) work?

The protocol for RRBS is described in the steps below. 

1. Digestion: Genomic DNA is cut into smaller, sequence-specific fragments using a restriction enzyme, typically MspI.
2. End Repair & Adapter Ligation: Following digestion, the DNA fragments (5’CG overhangs) undergo end repair to fix any breaks and A-tails are added on. Adapter ligation follows, where methylated adapter oligos are added to the DNA ends to prevent conversion of cytosines to uracils during bisulfite treatment.
3. Size Selection: The ligated DNA fragments are loaded onto an agarose gel, and fragments of desired sizes (e.g., 40-120 bp and 120-220 bp) are isolated for further processing. The specific size ranges may vary depending on the protocol used.
4. Bisulfite Conversion: Bisulfite treatment is applied to the isolated DNA fragments, which converts unmethylated cytosines to uracils while leaving methylated cytosines unchanged.
5. PCR Amplification: The bisulfite-treated DNA fragments are amplified via PCR to increase their abundance for subsequent sequencing.
6. Sequencing: The amplified DNA fragments are sequenced using next-generation sequencing technology, commonly on the Illumina platform.
7. Bioinformatics: Following sequencing, bioinformatics analysis is carried out to align the sequencing reads to a reference genome and identify methylated cytosines.