Bisulfite Sequencing Using Small DNA Amounts.

Methods in molecular biology (Clifton, N.J.) (2017-10-21)
Susanne Edelmann, Stefan Scholten
ABSTRACT

Bisulfite sequencing (BS-seq) enables the detection of DNA methylation at cytosine residues (5mC) at single-nucleotide resolution. For many applications, a limiting factor of conventional BS-seq protocols is the high amount of DNA required, since the treatment with bisulfite causes severe DNA fragmentation. Here, we describe a post-bisulfite tagging method that accounts for this problem. Illumina-compatible BS-seq libraries can be obtained from as little as five single haploid maize cells, enabling whole genome BS-seq (WGBS) for the generation of genome-wide, cell-type specific DNA methylation profiles. The method can also be used to analyze defined fractions of genomes from limited samples by Reduced Representation Bisulfite Sequencing (RRBS). This involves restriction digestion, gel separation and fragment elution prior to BS-seq library preparation to enrich certain areas of the genome. This reduction of represented genomic regions lowers the sequencing cost considerably while providing an accurate assessment of total genome-wide DNA methylation levels and assessment of DNA methylation in categorical genomic regions.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Imprint® DNA Modification Kit, For bisulfite DNA conversion & purification