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gmassacc 04-16-2020 02:36 PM

RNA-Seq Library Prep from PAXgene RNA Blood Tubes
Hi all,

My group is starting an RNA-seq project from patient frozen blood stored in PAXgene RNA blood collection tubes. We have experience performing library preps on PBMCs and isolated monocytes using the Takara Bio SMARTer RNA-seq kits. For this project, we have heard that one issue that could arise is the globin gene counts after sequencing. Has anybody had any issues with this?

Right now, I am thinking about extracting RNA from the PAXgene tube using the QIAGEN PAXgene extraction kit, then using this kit: GLOBINclear Kit for globin mRNA depletion and then running the standard library prep kits we have in the past.

However, Illumina sells a library prep kit that removes globin mRNA in the library prep itself:

Does anybody have any experience with this kit?

Thanks for all your help!

luc 04-16-2020 04:20 PM

A secret tip:
Much easier than any other solution.

nucacidhunter 04-17-2020 01:23 AM

Illumina kit is gold standard in this application if RNA quantity is not limiting. On the other hand performance of Qiagen kit varies and I would suggest to trial it before committing to use it.

jteeee2 04-17-2020 06:29 AM

Agree with @nucacidhunter. We were initially excited about the new Qiagen product but have also seen some odd performance issues...particularly when including globin-reduction probes. The Illumina TruSeq kit is solid. I've had good success with the NEBNext Ultra II Directional RNA kits with globin depletion. NEB also has a protocol for globin depletion followed by poly-A enrichment if you were interested in limiting transcript coverage to mRNAs.

gmassacc 04-17-2020 07:36 AM

Thanks for the replies! I will look into the NEB kit and the Illumina!

jteeee2 04-17-2020 10:38 AM

There's also this paper that came out a few days ago which suggests you don't need to deplete globin at all if you're starting with Paxgene blood.

I'd probably err on the side of caution and go ahead with your depletion strategy:) It won't hurt.

gmassacc 04-20-2020 07:04 AM

Super informative paper! Thanks jteeee2!

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