crustacean small RNA library QC

[fbarreto]

Hi, everyone,

We're trying for the first time to prepare small RNA libraries for our crustacean model, and we're using the NEBNext small RNA kit. We just finished running the PCR products (before size selection) on BioAnalyzer. The trace looks very different than what I expected, but I only have the kit manual as reference. Mostly, I am confused by the very strong fragment at ~288 bp. The small RNAs are expected to be enriched at ~143-147, so my guess is that the peak at 150 is what I want.

Has anyone seen this? The concentration of the 150 peak is high enough for sequencing. So I am assuming I can just proceed with size-selection of that peak and be fine, unless someone has experienced this pattern and can comment whether my libraries failed and should not be sequenced.

Thanks for any feedback!

[nucacidhunter]

150 bp is the target and it looks fine. The larger peaks depend on presence of other RNA species and would not be a concern. You might be able to correlate them with input RNA peaks if you have run a small RNA Chip.

[fbarreto]

Thank you for your feedback! Looking at the TruSeq small RNA guide, it seems this is a common possibility, and may vary with organisms. I will proceed with size-selection.

[sbarberan]

How many cycles of PCR did you perform? We have seen a big peak ~280 when you over amplify libraries.

I think that these products at 280 could be 'bulge' products generated when you deplete primers, see (http://www.lsi.umich.edu/files/SmallRNACloning.pdf) and search for bulge.

[fbarreto]

Hi, sbarberan,

We did 14 PCR cycles, with total starting RNA of ~700 ng. In consultation with our local sequencing core who does small RNA preps, they suggest these look fine, and we're proceeding with PAGE size-selection of the 140-160 bp bands. Fingers crossed.

I will update the thread when I get new BioA traces or sequences.

Thanks!

[sbarberan]

I'm sure if you PAGE size-select the libraries will be fine to sequence.

For next time I highly recommend that you use a gel-free library preparation kit. I work for Somagenics, and we just released our own small RNA library preparation kit that is gel-free and highly accurate (https://somagenics.com/realseq-ac). But there are other companies that also have gel-free library preparation kits for small RNAs.