Dinh:OxBS Protocol

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Revision as of 22:14, 19 December 2014 by >Dinh
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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. The most important component in the kit in the oxidant reagent. 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:

Preparation of the sequencing and digestion spike-in controls

  • The duplexed 100 bp control DNA is provided in the TrueMethyl kit. We do not need to order this oligo ourselves, however, the design of the oligo contains 5mC in many positions, which would be expensive to synthesize
    • 1 = 5hmC, 5 = 5mC
    • FWD:5’5T5A5CCA5AAC5A5AAA5ATA5GATCA5GGCGAAT5CGA[T1GA]ATCAGTT55GCG5TTTACGAAGTGCGA5AGC5TTAGTGATGTGATGGGTGGTATGG 3’
    • Sequence pre-conversion :5’ CTCACCCACAACCACAAACATACGATCACGGCGAATCCGA[TCGA]ATCAGTTCCGCGCTTTACGAAGTGCGACAGCCTTAGTGATGTGATGGGTGGTATGG 3’
    • Sequence post-conversion:5’ CTCACTTACAATCACAAACATACGATTACGGTGAATCTGA[TTGA]ATTAGTTCCGTGCTTTATGAAGTGTGACAGTCTTAGTGATGTGATGGGTGGTATGG 3’
    • Methylated Forward primer: 5T5A5CCA5AAC5A5AAA5A
    • Unmethylated Forward primer: TTTATTTATAATTATAAATA
  • I plan to generate copies of this control DNA, we need to amplify with dhmCTP/dATP/dTTP/dGTP mixture. This would add 5hmC's in all cytosine positions on the forward strand except for the portions corresponding to the primers. Some cytosines in the CG contexts in the Forward primer can be methylated with MsssI.