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

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

Published on: February 24, 2015

Generating Sequencing-Based DNA Methylation Maps from Low DNA Input Samples.

Suzan Al Momani^1,2, Euan J Rodger^1,2, Peter A Stockwell¹

¹Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand.

Methods in Molecular Biology (Clifton, N.J.)

|February 1, 2022

Summary

This summary is machine-generated.

This study presents an optimized protocol for reduced representation bisulfite sequencing (RRBS-seq) library preparation. The enhanced method efficiently analyzes DNA methylation patterns from low input DNA amounts, including formalin-fixed tissues.

Keywords:

CpG island DMAP DNA methylation Epigenetics FFPE Methylome Multiplexed Reduced representation bisulfite sequencing

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Area of Science:

Epigenetics
Genomics
Molecular Biology

Background:

Reduced representation bisulfite sequencing (RRBS) targets CpG-rich regions for DNA methylation analysis.
RRBS offers cost-effective genome-wide methylation profiling with high coverage depth.
Focusing on regulatory elements like promoters and enhancers is key for epigenetic studies.

Purpose of the Study:

To describe a modified RRBS sequencing (RRBS-seq) library preparation protocol.
To optimize the protocol for low input DNA amounts (10-100 ng).
To enable single base-resolution DNA methylation analysis.

Main Methods:

Modified RRBS-seq library preparation protocol.
Inclusion of deparaffinization for formalin-fixed samples.
Replacement of gel size-selection with sample purification beads.
Protocol optimized for 10-100 ng DNA input.

Main Results:

Successful generation of single base-resolution DNA methylation libraries.
Robust and reproducible results obtained from various tissues and cells.
Protocol applicable to formalin-fixed tissues.
Protocol completion within 3 days.

Conclusions:

The modified RRBS-seq protocol is efficient for low DNA input.
This method provides high-resolution DNA methylation data.
The protocol is versatile, applicable to diverse sample types including preserved tissues.