1. DNA Methylation mapping
Reduced Representation Bisulfite Sequencing
(RRBS)
1. Dynamic DNA methylation across diverse human cell lines and tissues;
Katherine E. Varley et al, Genome Res : originally published online January 16, 2013.
2. Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling;
Hongcang Gu1, Zachary D Smith1–3, Christoph Bock1–4, Patrick Boyle1, Andreas Gnirke1 & Alexander Meissner1–3; Nature Protocols, VOL.6 NO.4 | 2011.
3. SOLiD™ Bisulfite-Converted Fragment Library Preparation Protocol.

2. Bisulfite conversion reaction
Bisulfite Sequencing
Bisulfite conversion reaction
cytosine → cytosine-6- sulfonate;
cytosine-6-sulfonate → uracil-6-sulfonate;
uracil-6-sulfonate → uracil.
5-methylcytosines is non-reactive to bisulfite ions - therefore remains unchanged.

3. Advantages:
- Quantitative method directly measures C/T percentage as all BS approaches.
- CpG enrichment: RRBS uniquely uses a specific restriction enzyme as Msp1 that cut in CpG rich areas.
- Decreases the amount of sequencing required as well as the cost relative to WGBS.
- Only a low sample concentration, between  ng, is required for accurate data analysis.
- Formalin-fixed and paraffin-embedded inputs can also be used.
- Allows for the sequencing of methylated areas that are unable to be properly profiled
using conventional bisulfite sequencing techniques.
- MethylC-seq and MeDip-seq have a greater genome-wide coverage of CpGs compared to RRBS,
but RRBS has a greater coverage on CpG islands is consider quantitative and has single CpG resolution.
- The data obtained on RRBS and the Illumina Infinium methylation are highly comparable
as both use an absolute measurement of DNA.
Limitations:
- Restriction Enzyme limitations: MspI digestion covers the majority,
but not all the CG regions in the genome. Most CpG island and promoters are covered.
- Non-proofreading polymerase must be used as a proof-reading enzyme would stop
at uracil residues found in the ssDNA template.
- Complete denaturation and conversion is a must as in all BS methods.

4. Flowchart of RRBS library construction for Solid
Genomic DNA isolation
Msp1 digestion (C/CGG): CpG islands enrichment
Filling in and A-taililing: to facilitate ligation of adapters
Adapter ligation: use 10x less than original protocol
Nick-translation: to fill the ligation gap and to replace C with metC
Gel sizing: select for fragments bp (plus the length of the
adapters); carrier DNA added if small amount od DNA
Bisulfite conversion: Zymo Research gold kit; 50o for 60min x 16 cycles
PCR amplification: test PCR to select number of cycles;
amplify the whole library
Sequencing: MGL
Analysis: MGL

5. Msp digestion and ligation
The top sequence of P1 is the one that is ordered with methylated Cs
For small amounts of DNA:
- 10 times less adapters
- ligation 20 hours at 16o.
MSP digestion of genomic DNA:
Msp repeats are detected
After ligation Nick Translation :
- to replace C with 5-metC
- covalent bond between adapter
and A on the insert.
From Hongcang Gu et al, ; Nature Protocols, VOL.6 NO.4 ,2011.

6. Size selection Bisulfite conversion: Zymo Research gold kit;
95 for 30”o, 50o for 60min x 16 cycles
Size selection on 2% agarose gel:
Cut fragments bp
Add sonicated dephosphorylated
e. coli DNA as carrier

7. Test PCR To minimize the bias that might be introduced by PCR
Cycles:
For final library amplification use the lowest number
that generates enough PCR products
200 ml reaction aliquote in 8 wells of 96 well plate.
After PCR combine all reactions and purify the library.

8. Profile of the libraries analyzed on Transgenomics HPLC
MW Markers

9. Profile of the libraries run on Bioanalyzer