Home › Cell Biology › Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution
Cell Biology JoVE (Open Access) Citable · DOI

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DOI: 10.3791/52246-v
What you'll learn
  • ✓ Perform adapter ligation and size-select DNA fragments via gel purification
  • ✓ Execute bisulfite conversion and enrichment PCR for methylation analysis
  • ✓ Prepare sequencing libraries and interpret base pair resolution CpG methylation data
Protocol

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

Difficulty
advanced
Total time
~2–3 days (including bisulfite conversion and PCR cycles)
Biosafety
BSL-1

Steps

1
Ligate adapters and size-select DNA via gel purification

Perform restriction enzyme digestion and adapter ligation to DNA, then purify size-selected fragments by gel electrophoresis. This step prepares genomic DNA for methylation analysis.

▶ 02:22
2
Convert bisulfite, enrich, and purify library DNA

Treat adapter-ligated DNA with bisulfite to convert unmethylated cytosines, then perform enrichment PCR and purify the resulting product. This generates methylation-specific sequencing templates.

▶ 07:32
3
Prepare final library for sequencing

Complete final processing steps to generate sequencing-ready library from purified bisulfite-converted DNA. The library is now ready for loading onto a sequencer.

▶ 09:40
4
Analyze CpG methylation patterns and interpret results

Review sequencing output to identify base pair-level DNA methylation profiles at GC-rich genomic regions. This step demonstrates data quality and methylation calling from ERRBS analysis.

▶ 10:42
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