RNA-Seq Library Prep from PAXgene RNA Blood Tubes

[gmassacc]

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: https://www.illumina.com/products/by...na-globin.html

Does anybody have any experience with this kit?

Thanks for all your help!

[luc]

A secret tip:

https://www.qiagen.com/us/products/discovery-and-translational-research/next-generation-sequencing/rna-sequencing/ribosomal-rna-and-globin-mrna-removal/qiaseq-fastselect-rrna-hmr-kits/#orderinginformation

Much easier than any other solution.

[nucacidhunter]

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]

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]

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

[jteeee2]

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.

https://www.nature.com/articles/s41598-020-62801-6

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

[gmassacc]

Super informative paper! Thanks jteeee2!

[LJCatg]

GLOBINclear effectiveness

Hi gmassacc, did you end up going with the GLOBINclear kit in the end?

I'm using it for a project and have been having some difficulties getting it to work to a satisfactory level consistently. I'm intending to run it as past of an mRNA sequencing protocol from PAXgene blood RNA using the TruSeq mRNA library prep kit.

The main problem I'm encountering is when the wash solution is added, the bead pellet just refuses to disperse at all. I thought perhaps I could be leaving it dry for too long after I aspirate the binding buffer, but this doesn't seem to be the case as it happens even when I run 2 samples.

The end result is me losing 75-80% of my precious RNA, and getting poor OD ratios alongside it, mainly indicating salt contamination. I am very careful to remove as much wash buffer as possible but the salt still remains

I have previously ran this kit on very similar samples and had no problems, excellent recovery rate and little to no reduction in RINe.

Do you or anyone else have any tips on how to get this kit to work consistently?

Thanks in advance!