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  • mboth
    Member
    • Oct 2010
    • 23

    #1

    Any advice on RNA bisulfite sequencing?

    Hi everyone,
    We are planning to treat total RNA (rRNA removed) with bisulfite and convert the nonmethylated cytosines to uracil. This is a harsh treatment and will brake up mRNA into very small fragments.
    We will then use the TruSeq small RNA kit to produce the library. Thus we are expecting to be able to sequence all sorts of small RNAs, together with the chopped up fragments of mRNA.
    Has anybody done bisulfite sequencing, and is able to provide advice or warn me of any pitfalls?
    Thanks a lot, I appreciate any comment.
    Maike
  • mboth
    Member
    • Oct 2010
    • 23

    #2
    On second thoughts, it seems that the ends of RNA are being modified when submitted to bisulfite/alkali treatment, forming a 2′,3′ cyclic phosphate (J. Am. Chem. Soc., 1999, 121 (23), pp 5364–5372), so that the TruSeq adapters are unlikely to ligate.
    Has anybody got any idea how to reverse this? Is there a commercial 2′,3′ cyclic nucleotide phosphodiesterase?
    My biochemistry is not very good, so again any comment is very welcome!
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