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  • Low input directional viral RNA sequencing

    Dear users,

    I would like to use directional RNA sequencing protocol (with TrueSeq siRNA adapters) to prepare libraries from fragmented RNA of purified virus. After the fragmentation and end repair I`ve got only around 1 ng of RNA (depending on Bioanalyzer quantification).

    So, the question is: do I have chances to get a library from such a low input and does someone have experinces with low input in directional RNA sequencing library preparation? Would I need to adjust (lower) the concentration of adapters? I would be happy for any remarks.

    Thank you very much!

  • #2
    The company I work for routinely deals with RNA of low input. We've had moderate success generating libraries with as little as 1ng. However, you will end up having a lot of adapter dimer formation. From my experience, you should not lower the adapter concentration as it will reduce the efficiency of the ligation reactions. Instead, run a gel after the PCR step to remove the adapter dimer bands.

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    • #3
      We have a robot that will go down to 250pg so if you are interested in our services, please send me a message.

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      • #4
        Thank you,

        kcchan: you`ve done it for the directional RNA sequencing protocol?
        Anyway I have to cut the library band from the gel in the last step, my fear is only that I would not be able to see any band with such a low input... have you tried to cut invisible bands?

        Thank you.

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