Pippin Prep vs. gel elution for RNA-Seq

[shurjo]

I had a chance to do a side-by-side comparison of Pippin Prep (PP) and agarose gels (AG) for size selection during RNA-Seq library prep and would like to share these results with the community and get some feedback.

In the picture below, I show eight RNA-Seq libraries run on a BioAnalyzer HS DNA chip. The four sharp and high peaks are from PP while the four slightly lower and broader peaks are from AG. Equal aliquots of four adapter-ligated cDNAs were used for both platforms (so it's really 4 samples, then two techniques on each sample). Noticeably, the GS libraries have a lot more non-target material both above and below the peak, although all eight have a "bump" close to the upper size marker at 10380bp. A question: which of these would make the better candidate for submission to the sequencing center? I am leaning towards the PP as there is lesser background. Any advice from folks who are routinely using Pippin Prep for RNA-Seq?

Thanks,

Shurjo

[mbk0asis]

No way to remove adapter dimers?

The quote below is an old post from here in Seqanswers.
This guy compared two library size selection methods, agarose gel elution and pippin prep with his samples.
I like the sharp and narrow results of pippin prep, but adapter dimers still appeared at ~120bp. I thought most dimers would disappear when used pippin prep.
Has anyone tried pippin prep? Would somebody tell me how good it is?
And I'm planing to size select out 200~500bp. Would a broad range elution affect performance of the machine?

Thank you!!

I had a chance to do a side-by-side comparison of Pippin Prep (PP) and agarose gels (AG) for size selection during RNA-Seq library prep and would like to share these results with the community and get some feedback.

In the picture below, I show eight RNA-Seq libraries run on a BioAnalyzer HS DNA chip. The four sharp and high peaks are from PP while the four slightly lower and broader peaks are from AG. Equal aliquots of four adapter-ligated cDNAs were used for both platforms (so it's really 4 samples, then two techniques on each sample). Noticeably, the GS libraries have a lot more non-target material both above and below the peak, although all eight have a "bump" close to the upper size marker at 10380bp. A question: which of these would make the better candidate for submission to the sequencing center? I am leaning towards the PP as there is lesser background. Any advice from folks who are routinely using Pippin Prep for RNA-Seq?

Thanks,

Shurjo

[luc]

The Pippen prep works just fine and reliable in our experience, but so can the gel extraction protocols. I would not think that the GE results from the previous post are representative. GE results can look a lot better - one might dissolve the gel pieces better at RT than at 50 degrees as most GE kit manufacturers recommend. Adapter dimers can also be removed with Ampure or equivalent beads. I don't see any signs of adapter dimer problems in the PDF above however.
If selecting a wide size range there can be potentially be problems with the collection well overflowing with the PP - I have not tried it, though - perhaps better use a low % agarose cassette then and ask their tech support?

The quote below is an old post from here in Seqanswers.
This guy compared two library size selection methods, agarose gel elution and pippin prep with his samples.
I like the sharp and narrow results of pippin prep, but adapter dimers still appeared at ~120bp. I thought most dimers would disappear when used pippin prep.
Has anyone tried pippin prep? Would somebody tell me how good it is?
And I'm planing to size select out 200~500bp. Would a broad range elution affect performance of the machine?

Thank you!!