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TruSeq RNA Library Preparation Kit V2 using one-third the reagents

It seems ridiculous to use the amount of reagents Illumina is recommending in their protocol as I am not sure what kid of use I could ever have for 30 μl of library at a concentration of 100+ nM, so I divided everything by three so I can get 150 libraries out of the kit.  The protocol is here:

TruSeq RNA Library Preparation Kit V2 using one-third the reagents

  1. Teresa permalink

    Hi Ethan,

    I really appreciate the time you’ve spent sharing your info. I’m about to start a large MiSeq project screening for RNA viruses within our native bees up here in Canada, but was wondering if you had a feel for how stable the products are at the given “safe stop points”. I am starting with TruSeq Stranded RNA sample prep protocol (LS so small scale). I would like to generate a small set of libraries to pool (say 10-12) for the first MiSeq run, and would prefer to trouble-shoot by starting with say 1-2 libraries. The protocols consistently state stop points up to 7 days, yet my experience leaves me to believe they should be stable at -20 for considerably longer. Do you have any feedback as to how long one can wait between cDNA, adding adapters and finally pooling to run for sequencing?

  2. ethanomics permalink

    Hi Terersa,
    I usually try to make through the adapter ligation step on the first day. Freeze-thaw cycles can denature short pieces of DNA and given the complexity of the library they won’t find their partner again, so in theory this could cause some biases. After the adapter ligation step the products are stable indefinitely at -20˚C after any of the AMPure purifications. It’s purified DNA in 10 mM Tris-pH 8.0. If you want some bee-specific advice on RNA-seq, a guy (Mat Welch) in our group is doing it. You could ask him some questions. His e-mail is on the UWA Plant Energy Biology web page.


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