Low input directional viral RNA sequencing

[CottonJack]

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!

[kcchan]

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.

[kwaraska]

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

[CottonJack]

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.