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  • Effects of total RNA fragmentation on small RNAs

    Hello,
    I want to sequence enriched RNA (it will include both mRNAs and bacterial small non-coding RNAs (<200 nt)). I would like to fragment long RNAs to ~100-200 nt fragments before library preparation however I am afraid that small RNAs will be fragmented too and I will lose it after cutting required length RNAs from the gel (I am planning to cut 150-350 bp fragments already with adapters). My fragmentation buffer is 10 mM Tris-HCl (pH 7.4) and 10 mM ZnCl2, reaction conditions: 75 °C for 2.5 min.
    RNAs I want to sequence are not abundant - every loss of RNA sample should be avoided!
    Maybe someone has experience with the fragmentation of total RNA? Does it affect small RNAs, is it severely fragmented? Would it be better for me to prepare different libraries - one for small RNAs, another - for long RNAs?
    Thank you for your time and answer!

  • #2
    I assume chemical fragmentation will affect the small fragments to the same degree as the big ones.
    If you have an opportunity to sonicate samples? Small fragments are pretty safe from shearing.

    Another way to achieve that might be size selection (SPRI-beads) and separate fragmentation of the large fragments. Obviously, this will entail losses and add complexity.

    You might want to have a look at the Biorad Sequoia and Diagenode D-plex library prep kits for both short and long RNAs.

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