RRBS was performed according to a previously published protocol (Boyle et al. 2012 (link)). Briefly, genomic DNA was isolated using the PureLink genomic DNA kit (Invitrogen) according to the manufacturer's instructions and subjected to enzymatic digestion by MSPI (New England Biolabs) for 16 h. Klenow polymerase (New England Biolabs) was then added to each sample for end filling and poly-A tailing followed by TruSeq adapter ligation (Illumina). The library was then pooled and subjected to either bisulfite treatment as in the RRBS protocol or to an OX-RRBS protocol using the CEGX kit (Cambridge Epigenetix). Validation of oxidation efficiency was done with the provided DNA sequences that were also A-tailed and adapter-ligated. The sequence data from this study have been submitted to Gene Expression Omnibus under accession no. GSE60209.
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RRBS Protocol for Genome-Wide DNA Methylation
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Variable analysis
independent variables
- Enzymatic digestion by MSPI (New England Biolabs) for 16 h
- Bisulfite treatment as in the RRBS protocol
- OX-RRBS protocol using the CEGX kit (Cambridge Epigenetix)
- Sequence data from this study
- Genomic DNA isolated using the PureLink genomic DNA kit (Invitrogen) according to the manufacturer's instructions
- Klenow polymerase (New England Biolabs) for end filling and poly-A tailing
- TruSeq adapter ligation (Illumina)
- Validation of oxidation efficiency with the provided DNA sequences that were also A-tailed and adapter-ligated
- Positive control: Provided DNA sequences that were A-tailed and adapter-ligated
- Negative control: Not explicitly mentioned
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