Dinh:OxBS Protocol
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OxBS protocol
- Cambridge Epigenetix (CEGX) TrueMethyl kit: although each reaction costs > $100, they can have maximum input of only about 1 ug per reaction provided in the kit. To get enough input for BSPP, we need to use at least 2 reactions from the kit. After working with the kit once with Tiffany, I decided that it would be best to invest in purchasing individual components of the TrueMethyl kit & then making other components ourselves. Since the oxidant reagent is simply a solution of KRuO4 and NaOH we can make this solution & always use a control DNA to quantify the efficiency of conversion.
- References & notes:
- Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine [Nature Protocols] http://www.nature.com/nprot/journal/v8/n10/full/nprot.2013.115.html
- Supplementary Procedures from Booth et al [Science] http://www.sciencemag.org/content/suppl/2012/04/25/science.1220671.DC1/Booth-SOM.revision.1.pdf
- TrueMethyl kit protocols
Preparation of the sequencing and digestion spike-in controls
- The 100 bp control DNA is provided in the TrueMethyl kit.